N-(4-hydroxy-4-methyl-cyclohexyl)-4-phenyl-benzenesulfonamides and N-(4-hydroxy-4-methyl-cyclohexyl)-4-(2-pyridyl)benzenesulfonamides and their therapeutic use

ABSTRACT

The present invention pertains generally to the field of therapeutic compounds. More specifically the present invention pertains to certain substituted N-(4-hydroxy-4-methyl-cyclohexyl)-4-phenyl-benzenesulfonamide and N-(4-hydroxy-4-methyl-cyclohexyl)-4-(2-pyridyl)benzenesulfonamide compounds (collectively referred to herein as HMC compounds) that are useful, for example, in the treatment of disorders (e.g., diseases) including, inflammation and/or joint destruction and/or bone loss; disorders mediated by excessive and/or inappropriate and/or prolonged activation of the immune system; inflammatory and autoimmune disorders, for example, rheumatoid arthritis; psoriasis; psoriatic arthritis; chronic obstructive pulmonary disease (COPD); asthma; atherosclerosis; inflammatory bowel disease; ankylosing spondylitis; multiple sclerosis; systemic lupus erythematosus; Sjogren&#39;s syndrome; a disorder associated with bone loss, such as bone loss associated with excessive osteoclast activity in rheumatoid arthritis, osteoporosis, cancer-associated bone disease, or Paget&#39;s disease; cancer, such as a haematological malignancy, such as multiple myeloma, leukemia, or lymphoma, or a solid tumor cancer, such as bladder cancer, breast cancer (female and/or male), colon cancer, renal cell carcinoma, kidney cancer, lung cancer, pancreatic cancer, gastric cancer, prostate cancer, brain cancer, skin cancer, thyroid cancer, basal cell ameloblastoma, or melanoma; a disorder associated with fibrosis, such as systemic sclerosis or scleroderma; or a rare vasculitide, such as Behçet&#39;s disease. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, for example, in therapy.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/899,422, filed Dec. 17, 2015. U.S. application Ser. No. 14/899,422 isa 35 U.S.C. §371 national phase application of International ApplicationSerial No. PCT/GB2014/051921, filed Jun. 24, 2014 (WO 2014/207445).International Application Serial No. PCT/GB2014/051921 claims priorityto United Kingdom patent application number 1311361.8, filed Jun. 26,2013. The contents of each of these applications are incorporated hereinby reference in their entirety.

TECHNICAL FIELD

The present invention pertains generally to the field of therapeuticcompounds. More specifically the present invention pertains to certainsubstitutedN-(4-hydroxy-4-methyl-cyclohexyl)-4-phenyl-benzenesulfonamide andN-(4-hydroxy-4-methyl-cyclohexyl)-4-(2-pyridyl)benzenesulfonamidecompounds (collectively referred to herein as HMC compounds) that areuseful, for example, in the treatment of disorders (e.g., diseases)including, inflammation and/or joint destruction and/or bone loss;disorders mediated by excessive and/or inappropriate and/or prolongedactivation of the immune system; inflammatory and autoimmune disorders,for example, rheumatoid arthritis; psoriasis; psoriatic arthritis;chronic obstructive pulmonary disease (COPD); asthma; atherosclerosis;inflammatory bowel disease; ankylosing spondylitis; multiple sclerosis;systemic lupus erythematosus; Sjogren's syndrome; a disorder associatedwith bone loss, such as bone loss associated with excessive osteoclastactivity in rheumatoid arthritis, osteoporosis, cancer-associated bonedisease, or Paget's disease; cancer, such as a haematologicalmalignancy, such as multiple myeloma, leukemia, or lymphoma, or a solidtumour cancer, such as bladder cancer, breast cancer (female and/ormale), colon cancer, renal cell carcinoma, kidney cancer, lung cancer,pancreatic cancer, gastric cancer, prostate cancer, brain cancer, skincancer, thyroid cancer, basal cell ameloblastoma, or melanoma; adisorder associated with fibrosis, such as systemic sclerosis orscleroderma; or a rare vasculitide, such as Behçet's disease. Thepresent invention also pertains to pharmaceutical compositionscomprising such compounds, and the use of such compounds andcompositions, for example, in therapy.

BACKGROUND

A number of publications are cited herein in order to more fullydescribe and disclose the invention and the state of the art to whichthe invention pertains. Each of these publications is incorporatedherein by reference in its entirety into the present disclosure, to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

This disclosure includes information that may be useful in understandingthe present invention. It is not an admission that any of theinformation provided herein is prior art or relevant to the presentlyclaimed invention, or that any publication specifically or implicitlyreferenced is prior art.

Chronic Inflammatory Disease

Inflammation is the immune response of tissues due to bodily injury.Acute inflammation is a normal, protective response that protects andheals the body following physical injury or infection, characterised byheat, swelling, and redness at the site of the injury.

However, if inflammation persists for a prolonged period, it becomeschronic. Chronic inflammation is a hallmark of, and a contributingfactor to, a range of disease conditions including rheumatoid arthritis,inflammatory bowel disease, systemic lupus erythematosus, multiplesclerosis and psoriasis.

The inflammatory process is complex and involves a biological cascade ofmolecular and cellular signals that alter physiological responses. Atthe site of the injury, cells release molecular signals such ascytokines and interleukins that cause a number of changes in theaffected area including dilation of blood vessels, increased blood flow,increased vascular permeability, invasion by leukocytes (white bloodcells), and exudation of fluids containing proteins like immunoglobulins(antibodies). Several different types of leukocytes, includinggranulocytes, monocytes, and lymphocytes, are involved in theinflammatory cascade. However, chronic inflammation is primarilymediated by monocytes and long-lived macrophages; monocytes mature intomacrophages once they leave the bloodstream and enter tissues.Macrophages engulf and digest microorganisms, foreign invaders, andsenescent cells and macrophages release several different chemicalmediators, including Tumour Necrosis Factor-alpha (TNFα), interleukins(e.g., IL-1, IL-6, IL-12 and IL-23) and prostaglandins that perpetuatethe inflammatory response. At later stages, other cells, includinglymphocytes, invade the affected tissues.

There is thus a common pathology underlying a wide variety of chronicinflammatory conditions. In addition, features of chronic inflammationare also observed in other diseases including cancer and metabolicdiseases such as obesity and diabetes.

One of the most common chronic inflammatory conditions is rheumatoidarthritis (RA), a condition which affects up to 2% of the populationworldwide. Although it is a complex disease, there are a number ofphysiological, cellular, and biochemical factors associated with theprogression of RA that are common to a range of other diseases,including those with a component of autoimmunity (e.g., multiplesclerosis), inflammation (e.g., atherosclerosis and cancer), bone loss(e.g., osteoporosis) and proliferation (e.g., haematologicalmalignancies). This makes the understanding of RA important not only forthe study of a much broader range of diseases, but also suggests thatpharmaceutical agents that work via modification of these commonprocesses may have utility beyond RA. The latter is borne out byclinical practice where RA drugs have been shown to have broad utilityacross a variety of other conditions.

Rheumatoid Arthritis and Related Autoimmune/Inflammatory Diseases

Rheumatoid arthritis (RA) is an autoimmune disorder characterized bychronic inflammation of the synovial lining of multiple joints coupledto progressive joint degradation. RA commonly affects the joints of thewrist and hands and may also affect the elbows, shoulders, hips, neckand knees leading to severe pain and disability (see, e.g., Scott etal., 2010). The World Health Organisation predicts that 23.7 millionpeople suffer from RA, with incidence rising due to the associationbetween the condition and increasing age.

The exact cause of RA, as for all the autoimmune disorders, remainsunclear, although possible triggers include reduced self-tolerance, anabnormal response to environmental factors, infectious agents, andhormonal stimulus (see, e.g., Klareskog et al., 2006; Firestein et al.,2005).

At the cellular level, development of RA usually commences with T-cellsinfiltrating the synovial membrane lining the affected joint; this thenleads to the activation of monocytes, macrophages and synovialfibroblasts by way of cell-cell contact and the subsequent release ofvarious cytokines, including tumour necrosis factor-alpha (TNFα) andpro-inflammatory interleukins such as IL-1, IL-6, IL-12 and IL-23 (see,e.g., Astry et al., 2011). These pro-inflammatory cytokines are theninstrumental in orchestrating several complex signal transductioncascades, including the NFκB, Interferon Regulatory Factor (IRF),Toll-like receptor (TLR), and Jak/STAT pathways (see, e.g., Malemud etal., 2010) which lead to the induction of genes coding for variousproducts that propagate the inflammatory response and also promotetissue destruction. These products include tissue-degrading enzymes suchas collagenases, matrix metalloproteinases (MMPs), cathepsins, and otherpro-inflammatory factors such as selectins, integrins, leukotrienes,prostaglandins, chemokines, and other cytokines (see, e.g., McInnes etal., 2007; Smolen et al., 2003). In addition, these cells also increasethe production of MMPs, leading to the degradation of the extra cellularmatrix and loss of cartilage within the joint (see, e.g., Sun, 2010), aprocess that also involves a specialised class of cells known asosteoclasts and a factor known as Receptor Activator of Nuclear FactorKappa-B Ligand (RANKL) (see, e.g., Takayanagi, 2009).

RANKL is an essential factor for the generation of osteoclasts, andupregulated RANKL-production leads to increased osteoclastdifferentiation and ultimately bone destruction (see, e.g., Long et al.,2012). The inflammatory response in RA leads to the accumulation oflymphocytes, dendritic cells, and macrophages, all operating locally toproduce cytokines and other pro-inflammatory mediators such as TNFα andIL-6 which further potentiate the effects of RANKL on bone destruction.In addition, the inflammatory cascade leads to the hyperplasia ofsynoviocytes (see, e.g., Takayanagi, 2009), which in turn leads to thethickening and vascularisation of the synovium into a destructive andaggressive tissue known as a pannus. The pannus contains bothosteoclasts, which destroy bone, and metalloproteinases, which areinvolved in the destruction of cartilage.

As such, the RANKL axis is critical to the progression and pathology ofRA as well as to the osteoimmune system (the interplay between theimmune and bone systems), which is central to the pathology of a numberof different disease conditions, described below.

The Role of TNFα in RA

The TNF superfamily of receptors and ligands plays a key role in thecausation of inflammation and associated local and systemic bone loss.TNFα is a powerful pro-inflammatory agent that regulates many facets ofmacrophage function. It is rapidly released after trauma, infection, orexposure to bacterial-derived LPS and has been shown to be one of themost abundant early mediators in inflamed tissue. Among its variousfunctions is its central role in orchestrating the production of apro-inflammatory cytokine cascade. In addition to pro-inflammatorycytokines, TNFα also increases lipid signal transduction mediators suchas prostaglandins. Based on these roles, TNFα has been proposed as acentral player in inflammatory cell activation and recruitment and issuggested to play a critical role in the development of many chronicinflammatory diseases including rheumatoid arthritis (see, e.g., Liu,2005; Feldmann et al., 2001; Brennan et al., 1996; Brennan et al.,1992). The importance of TNFα in RA is highlighted by the finding thatantibodies blocking TNFα can prevent inflammation in animal models ofRA, and that anti-TNFα therapy is currently the most effective treatmentfor RA (see, e.g., Pisetsky, 2012, and further detail provided below).

TNFα itself instigates a signalling cascade which leads to theactivation of the transcription factors NFκB and AP-1 (see, e.g.,Parameswaran et al., 2010). Binding of TNFα and IL-1 to their respectivereceptors leads to the recruitment of downstream signal transducerscalled TRAFs. Further kinases are recruited by the TRAFs, and theresulting kinase complex activates the MAP-kinase pathway, ultimatelyleading to activation of AP-1, and the phosphorylation of IκB kinase.IκB is the inhibitor of NFκB, which acts by preventing translocation ofNFκB to the nucleus. Phosphorylation of IκB by IκB kinase leads todegradation of IκB. Once IκB has been degraded, NFκB migrates to thenucleus, where it promotes transcription of anti-apoptotic genes, whichpromote survival of T- and B-cells, thereby prolonging the immuneresponse. This prolongation of the inflammatory response is central tothe chronic nature of RA. The importance of NFκB activation isdemonstrated by the fact that inhibition of NFκB activity by inhibitorypeptides can prevent arthritis in animal models of RA (see, e.g., Jimiet al., 2004).

Other Key Factors in Rheumatoid Arthritis

As described above, a number of factors in addition to TNFα and NFκB actto promote inflammation in RA and other chronic inflammatory diseases.Amongst these are IL-6 and the Interferon Regulatory Factors (IRFs).

Interleukin-6 (IL-6) is a pro-inflammatory cytokine whose levels areincreased upon activation of various immune system cells duringinflammation in RA, predominantly macrophages and T cells. It haspleiotropic effects in disease via its key role in the acute phaseresponse and is heavily involved in governing the transition from acuteto chronic inflammation. It does this by modifying the composition ofthe white blood cell infiltrate in the inflammatory space, moving itfrom neutrophils to monocyte/macrophages (see, e.g., Gabay, 2006). Inaddition, IL-6 exerts stimulatory effects on T- and B-cells, thusfavouring chronic inflammatory responses, as well as on osteoclasts,thus promoting the turnover of bone. These effects are involved in thepathology of a broad range of autoimmune/inflammatory diseases beyondRA, including systemic lupus erythematosus, atherosclerosis, psoriasis,psoriatic arthritis, asthma, chronic obstructive pulmonary disease(COPD), Sjogren's syndrome, atherosclerosis, and inflammatory boweldisease, as well as in cancers such as multiple myeloma and prostatecancer.

In addition, IL-6 has been implicated in diseases involving bone loss(e.g., osteoporosis), diseases mediated by fibrosis (e.g., systemicsclerosis), diabetes, transplant rejection, various cancers (including,e.g., multiple myeloma, lymphoma, prostate cancer), neurodegenerativediseases (e.g., Alzheimer's), psychiatric disorders (e.g., depression),and certain rare vasculitides (e.g., Behçet's disease). For a fullreview, see, e.g., Rincon, 2012.

The interferon regulatory factors (IRFs) consist of a family oftranscription factors with diverse functions in the transcriptionalregulation of cellular responses in health and diseases. IRFs commonlycontain a DNA-binding domain in the N-terminus, with most members alsocontaining a C-terminal IRF-associated domain that mediatesprotein-protein interactions. Ten IRFs and several virus-encoded IRFhomologs have been identified in mammals. IRFs are activated in responseto endogenous and microbial stimuli during an immune response, andselectively and cooperatively modulate the expression of key cytokineand transcription factors involved in a variety of inflammatoryprocesses. For example, stimulation of the receptor for bacteriallipopolysaccharide, TLR-4 activates a signalling cascade which activatesboth NFκB and IRF-5, whilst IRF-7 is activated by a process involvingthe STAT family of transcription factors, which are also, butindependently, activated by IL-6.

The activation of the IRFs leads to a number of downstream effectsincluding the specification of macrophage fate (see, e.g., Krausgruberet al., 2011), T helper cell differentiation (see, e.g., Zhang et al.,2012) and B-cell proliferation (see, e.g., Minamino et al., 2012). Thesediverse roles in disease are underlined by data from animal knockoutmodels which show, for example, reduced levels of IL-6 and TNFα inresponse to inflammatory stimuli (see e.g., Takaoka et al., 2005).

In addition to the biological roles of the IRFs described above, severalIRF family members have been genetically associated with predispositionto inflammatory conditions. For example, polymorphisms in IRF-3 andIRF-7 are associated with susceptibility to systemic lupus erythematosus(see, e.g., Akahoshi et al., 2008; Fu et al., 2011). In addition, IRF-5,which controls the fate of macrophages, is associated withsusceptibility to RA, systemic lupus erythematosus, Wegener'sGranulomatosis, Sjogren's syndrome, and systemic sclerosis (see, e.g.,Sharif et al., 2012; Hu et al., 2011).

Treatment of Rheumatoid Arthritis

Early therapies for RA focussed on controlling the symptoms of thedisease, mainly by reduction of inflammation, rather than retardingdisease progression. These drugs included NSAIDs such as aspirin,diclofenac, and naproxen. Inflammation was further controlled byglucocorticoids, and their combination with NSAIDs provided reasonablyeffective short-term control of the inflammation. More recently, a moreaggressive approach to treating RA has been introduced starting atdisease onset, using so-called disease-modifying anti-rheumatic drugs(DMARDs), which act to slow or even prevent disease progression. Theseinclude a number of older drugs, including gold salts; sulfasalazine;antimalarials such as hydroxychloroquine; D-penicillamine;immunosuppressants such as mycophenolic acid, azathioprine, cyclosporineA, tacrolimus and sirolimus; minocycline; leflunomide; and mostimportantly, methotrexate (see, e.g., Smolen et al., 2003).

Methotrexate is now the gold-standard therapy for clinical trialcomparisons, and is generally used in combination with newer therapies.It is effective in most patients but, in common with all of the aboveagents, has significant gastrointestinal side effects, which lead toroughly 50% of patients eventually having to cease treatment (see, e.g.,Mount et al., 2005). A further drawback of these older DMARDs is thelength of time taken for the drug to start acting, ranging from weekswith methotrexate, to months with gold salts. Whilst full remissionsonly occur in about a quarter of patients, for those showing no effectit is not generally possible to stop therapy without suffering the riskof a more violent disease rebound (see, e.g., Smolen et al., 2003).

In recent years, the treatment of RA has been revolutionised by theadvent of biological agents which target specific inflammatory pathways.Several biological agents are currently approved for use in RA includinganti-IL-6 and IL-1 biologics such as tocilizumab (Actemra®) and anakinra(Kineret®) (see, e.g., Scott et al., 2010). However, the first and mostimportant of the biological agents are the anti-tumour necrosis factor(anti-TNF) therapies.

Anti-TNFα therapies are the market-leading treatment for RA. A varietyof anti-TNFα agents are available including neutralising antibodies suchas infliximab (Remicade®; J&J and Schering Plough) and adalimumab(Humira®; Abbott), or decoy receptors such as etanercept (Enbrel®; Amgenand Wyeth), both of which represent validated and highly effectivetreatments for RA as well as other diseases such as Crohn's disease andpsoriasis. A number of other inflammatory and autoimmune disorders arealso being investigated as potential targets. Other approaches toblocking the action of TNFα include the pegylated anti-TNFα fragmentcertolizumab (Cimzia®, UCB). All of these therapies act, ultimately, toprevent the activation of the downstream effectors of TNFα describedabove, including NFκB. However, in spite of their market success, theanti-TNFα therapies suffer from a number of side-effects includingincreased risk of certain malignancies such as lymphoma and seriousinfections such as Legionella and Listeria, as well as increased risk ofheart failure, Hepatitis B reactivation, and demyelinating disease.

Finally, and most recently, a JAK kinase inhibitor, tofacitinib(Xeljanz®, Pfizer) has supplemented the range of RA treatments. However,tofacitinib suffers from a number of safety concerns including increasedrisk of serious infections as well as increased risk of gastrointestinalperforations, liver damage, and certain cancers, that are likely tolimit its use in man (see, e.g., O'Shea et al., 2013).

As such, there remains a need for new and improved therapies for RA andother inflammatory diseases with a particular focus on improved safety.

The Osteoimmune System and Bone Disorders

The osteoimmune system is term for the combined and related interplaybetween the immune system and the skeletal system.

Under normal physiological conditions, the skeletal system providessupport, mobility, protection for vital organs, and a mineral reservoirfor calcium and phosphate. In order to achieve and adapt to thesefunctions, the skeleton exists in a dynamic equilibrium characterized bycontinuous osteoclast-mediated bone resorption and osteoblast-mediatedbone deposition (see, e.g., Karsenty et al., 2002). This biologicalprocess has been termed bone “remodelling” and occurs in coupled fashionwith osteoblasts producing the key osteoclast differentiation factors,including RANKL, described above, and osteoclasts promoting boneformation by producing osteoblastic mediators as they degrade bone.

Both innate and adaptive immune cells exert effects on osteoclasts andosteoblasts through a variety of cell-surface and secreted mediators(see, e.g., Takayanagi, 2009). Activation of the RANKL receptor (RANK)on osteoclast precursors starts a cascade of transcriptional changeswhich results in the formation of osteoclasts and the expression of themachinery needed for bone resorption including molecules needed forattachment to bone, acid secretion, and proteolysis. Many of thetranscription factors important for osteoclast differentiation are keyregulators of immune responses, such as NFκB and nuclear factor ofactivated T cells c1 (NFATc1) and this process is also potentiated byfactors involved in inflammation such as TNFα and IL-6.

In addition to its critical role in the progression and pathogenesis ofRA, the osteoimmune system plays a critical role in a number of otherdiseases including osteoporosis and other bone disorders and cancer(see, e.g., Dallas et al., 2011).

Osteoporosis is a common disease characterised by reduced bone density,deterioration of bone tissue, and an increased risk of fracture. Manyfactors contribute to the pathogenesis of osteoporosis including poordiet, lack of exercise, smoking, and excessive alcohol intake.Osteoporosis also arises in association with inflammatory diseases suchas rheumatoid arthritis, endocrine diseases such as thyrotoxicosis, andwith certain drug treatments such as treatment with glucocorticoids.Indeed, osteoporosis-related fragility fractures represent one of themost important complications that may occur in patients with rheumaticdiseases such as RA, systemic lupus erythematosus, and ankylosingspondylitis.

Paget's disease of bone is a common condition of unknown cause,characterised by increased bone turnover and disorganised boneremodelling, with areas of increased osteoclastic and osteoblastactivity. Although Pagetic bone is often denser than normal, theabnormal architecture causes the bone to be mechanically weak, resultingin bone deformity and increased susceptibility to pathological fracture.

IL-6, TNFα, and RANKL signalling have been shown to play a major role inosteoclast over-activity and a consequent increase in bone loss (see,e.g., Tanaka et al., 2003; Roodman, 2006). The use of drugs which affectthese pathways have been validated by the completion of clinical trialsof the monoclonal antibody against RANKL, AMG-162 (Denosumab®, Amgen),for the treatment of osteoporosis/multiple myeloma, as well as by anincreasing body of evidence that shows that the anti-TNFα and anti-IL-6therapies also prevent bone loss in arthritic diseases (see, e.g., Ogataet al., 2012; Billau, 2010).

The Osteoimmune System and Cancer

Many types of cancer affect bone. Cancer-associated bone disease can bemanifest by the occurrence of hypercalcaemia or the development ofosteolytic and/or osteosclerotic metastases. Increased osteoclastic boneresorption plays a key role in the pathogenesis of both conditions.Whilst almost any cancer can be complicated by bone metastases, the mostcommon sources are multiple myeloma, breast carcinoma, and prostatecarcinoma. The most common tumours associated with hypercalcaemia aremultiple myeloma, breast carcinoma, and lung carcinoma.

As described above, RANK/RANKL signalling is essential for osteoclastformation and bone resorption that occurs during skeletal remodelling.While physiological levels of RANK/RANKL signalling stimulate theproliferation and cell survival of mammary epithelial cells, aberrantRANK/RANKL signalling in these tissues has recently been shown toinfluence the onset and progression of breast tumorigenesis and blockingRANKL signalling using denosumab (Xgeva®, Amgen) has been shown to be aneffective in preventing the secondary complications of bone metastases,such as pathologic fracture, and hypercalcaemia in patients with breastcancer (see, e.g., Steger et al., 2011).

Therapies that block RANK/RANKL signalling may also decrease the abilityof osteotropic cancers to metastasize to bone. Signalling through RANKon the surface of human epithelial tumour cells as well as melanomacells has been shown to induce a chemotactic response in these tumourcells whilst in a murine model of melanoma metastasis, therapeutictreatment of mice with osteoprotegrin, which neutralizes the RANKLreceptor, RANK, significantly reduced tumour burden within the bones butnot other organs.

In addition to a role for RANKL in cancer, there is growing evidencethat activation of NFκB via molecules such as TNFα can play a major rolein the promotion and progression of both haematological malignancies,such as myeloma and lymphomas, and solid tumours, such as breast,prostate, and lung cancer (see, e.g., Baud et al., 2009). There is alsorising awareness of the role and importance of inflammation and theosteoimmune system in cancer and in the development of resistance toradiotherapy and to chemotherapeutic agents. Furthermore, it has beensuggested that inflammation is in fact one of the basic hallmarks ofcancer (see, e.g., Mantovani, 2009). Improving the efficacy ofanti-cancer treatments by prevention of NFκB activation is therefore apromising strategy to augment existing therapeutic regimes and iscurrently under investigation, most notably for the treatment ofmultiple myeloma.

Defects in the normal apoptotic pathways are also implicated in thedevelopment and progression of tumour cell growth as well as ininflammation. Apoptosis (programmed cell death) plays a key role in theremoval of abnormal cells; defects in the signalling cascades, whichwould normally lead to its induction, play a key role in oncogenesis.Radiotherapy and many chemotherapeutic agents act by causing cellulardamage, which would normally induce apoptosis; defects in the pathwaywill therefore also reduce the effectiveness of such agents. The mostimportant effector molecules in the signalling pathway leading toapoptosis are known as the caspases, which may be triggered by a numberof stimuli, including TNFα binding to its receptor. Mutations in thegenes which encode for the caspases have been found in a number oftumour types, including gastric, breast, renal cell, and cervicalcancers as well as commonly in T-cell lymphoblastic lymphoma and basalcell ameloblastomas (see, e.g., Philchenkov et al., 2004). Compoundswhich activate caspases, and thus sensitise cells to apoptosis, would behighly effective as cancer therapies either as single agents or inenhancing the effectiveness of existing cancer chemotherapy andradiotherapy.

Agents that Prevent Inflammation Disrupt the Osteoimmune System

The inventors have identified new compounds which, for example, preventinflammation and/or bone loss, and thus may be used in the treatment ofdiseases with an inflammatory or autoimmune component, including, forexample, rheumatoid arthritis, inflammatory bowel disease, systemiclupus erythematosus, atherosclerosis, asthma, chronic obstructivepulmonary disease (COPD), uveitis, pelvic inflammatory disease,endometriosis, psoriasis and psoriatic arthritis; diseases which involvebone loss, including, for example, bone loss associated with rheumatoidarthritis, osteoporosis, Paget's disease of bone, and multiple myeloma;as well as cancer associated with activation of NFκB, with aberrant NFκBsignalling, or with inflammation or IL-6 overproduction, includinghaematological malignancies such as multiple myeloma, leukaemia, T-celllymphoblastic lymphoma, and other lymphomas (e.g., non-Hodgkin'sLymphoma), and solid tumours such as bladder cancer, breast cancer(female and/or male), colon cancer, kidney cancer, lung cancer,pancreatic cancer, prostate cancer, brain cancer, skin cancer, thyroidcancer, and melanoma; cancer associated with the inactivation orimpairment of caspase-mediated cell death, such as gastric cancer,breast cancer, renal cancer, cervical cancer, and basal cellameloblastomas; conditions associated with modulated activity of IRF-5including Wegener's granulomatosis and systemic sclerosis; fibrosisassociated with overproduction of IL-6, such as systemic sclerosis orscleroderma; neurodegenerative diseases associated with IL-6overproduction, such as Alzheimer's disease; psychiatric disorders alsoassociated with IL-6 overproduction, such as depression; diseases ofangiogenesis associated with IL-6 overproduction such as age-relatedmacular degeneration and diabetic retinopathy, IL-6 associatedhyperplasias such as Castleman's disease and certain rare vasculitidesassociated with IL-6 overproduction, such as Behçet's disease.

Without wishing to be bound by any particular theory, the inventorsbelieve that this action may be via a mechanism that involves blockingTNFα, and/or RANKL-signalling and/or IRF activity and/or inhibition ofIL-6 production.

Known Compounds

Wang et al., 2010, describes certain compounds which apparently arehigh-affinity and selective dopamine D³ receptor full agonists. Examplesof compounds shown therein include the following (see, e.g., pages 18-19and 48-50 therein):

Chen et al., 2012 describes similar compounds.

Tsutsumi et al., 2005, describes certain compounds which apparently showDPP-IV inhibitory activity and apparently are useful in the treatment oftype II diabetes and obesity. The following compound is shown as Example89 on page 192 therein:

Hadida et al., 2007 describes certain compounds which allegedly areuseful as modulators of ATP-binding cassette (“ABC”) transporters orfragments thereof, including Cystic Fibrosis Transmembrane ConductanceRegulator (“CFTR”). The following compound is shown as Example 208 onpage 77 therein:

Ralston et al., 2005, describes certain biphenyl-4-sulfonic amides foruse: to inhibit osteoclast survival, formation, and/or activity; toinhibit conditions mediated by osteoclasts and/or characterised by boneresorption; in the treatment of bone disorders such as osteoporosis,rheumatoid arthritis, cancer associated bone disease, and Paget'sdisease; and in the treatment of conditions associated with inflammationor activation of the immune system. Examples of compounds shown thereininclude the following:

Greig et al., 2006, describes similar compounds.

Greig et al., 2008, describes certain biphenyl-4-sulfonic acid amidesfor the treatment of inflammation and/or joint destruction and/or boneloss; disorders mediated by excessive and/or inappropriate and/orprolonged activation of the immune system; inflammatory and autoimmunedisorders, for example, rheumatoid arthritis, psoriasis, psoriaticarthritis, chronic obstructive pulmonary disease (COPD),atherosclerosis, inflammatory bowel disease, and ankylosing spondylitis;and disorders associated with bone loss, such as bone loss associatedwith excessive osteoclast activity in rheumatoid arthritis,osteoporosis, cancer associated bone disease, and Paget's disease.Examples of compounds shown therein include the following:

Greig et al., 2010b, describes certain biphenyl-4-sulfonic acid amidesfor the treatment of inflammation and/or joint destruction and/or boneloss; disorders mediated by excessive and/or inappropriate and/orprolonged activation of the immune system; inflammatory and autoimmunedisorders, for example, rheumatoid arthritis, psoriasis, psoriaticarthritis, chronic obstructive pulmonary disease (COPD),atherosclerosis, inflammatory bowel disease, and ankylosing spondylitis;disorders associated with bone loss, such as bone loss associated withexcessive osteoclast activity in rheumatoid arthritis, osteoporosis,cancer-associated bone disease, and Paget's disease; and cancer, such asa haematological malignancy and a solid tumour. Examples of compoundsshown therein include the following:

Greig et al., 2013 describes similar compounds.

Greig et al., 2010a, describes certain biphenyl-4-sulfonic acid amidesfor the treatment of inflammation and/or joint destruction and/or boneloss; disorders mediated by excessive and/or inappropriate and/orprolonged activation of the immune system; inflammatory and autoimmunedisorders, for example, rheumatoid arthritis, psoriasis, psoriaticarthritis, chronic obstructive pulmonary disease (COPD),atherosclerosis, inflammatory bowel disease, and ankylosing spondylitis;disorders associated with bone loss, such as bone loss associated withexcessive osteoclast activity in rheumatoid arthritis, osteoporosis,cancer-associated bone disease, and Paget's disease; and cancer, such asa haematological malignancy and a solid tumour. Examples of compoundsshown therein include the following:

New Compounds with Improved Properties

The HMC compounds described herein are protected against several toxicliabilities that are present in the known compounds, especially thoseshown in Greig et al., 2010a and show improved efficacy in models ofdisease.

Without wishing to be bound to any particular theory, the inventorsbelieve that the particular combinations of substituents and theirpositions on the biaryl ring structure give rise to extraordinaryproperties. In addition to substantial improvements in acute in vivotoxicology, these combinations protect the compounds from generalcytotoxicity, genotoxicity, and cardiovascular safety liabilities seenin the known compounds. Specifically, the HMC compounds described hereinare negative for genotoxicity, show a substantial improvement in generalcytotoxicity, and are substantially protected against inhibition of thehuman Ether-à-go-go related gene (hERG), which represents a majorcardiovascular safety liability.

If a drug is to be used in the clinic, it must have a suitable safetyand efficacy profile. It must show adequate acute safety to allow dosingto humans without the expectation of serious general side-effects. Inaddition, it must not cause genetic damage (genotoxicity) because agentsthat are genotoxic can act as carcinogens in humans. A clinicallyacceptable drug should also not inhibit hERG, an ion-channel which, wheninhibited, can cause a fatal heart disorder known as long QT syndrome.Alongside these safety properties, the drug must be sufficiently potentagainst the biological target to give the desired therapeutic effect; itmust have a sufficient solubility to be absorbed from thegastrointestinal tract; and it must have sufficient stability to remainin the circulation long enough to reach the biological target.

The HMC compounds described herein demonstrate improved efficacy inmodels of rheumatoid arthritis, for example, as compared with thecompounds shown in Greig et al., 2010a. This is demonstrated both by agreater magnitude of effect on disease, as well as greater potency, bothof which are seen, importantly, when the HMC compounds are administeredwhen disease is already established. This mirrors the clinical settingfor the use of these compounds. Moreover these effects are seen withoutovert toxicity.

The reduction of toxicological properties (adverse effects) of a drug isa developmental barrier of equal challenge and importance as compared tothe optimization of pharmacodynamics (action of the drug on the body)and pharmacokinetic (action of the body on the drug) properties. The HMCcompounds described herein provide substantial advantages as oraltherapeutic agents (as compared to the known compounds) by improvingacute general in vivo toxicology, genotoxic and cytotoxic safety, andcardiovascular safety, with little or no change in in vivopharmacokinetics or loss of potency against the biological target.

The HMC compounds described herein combine the required characteristicsof orally active agents for the treatment of, for example, chronicinflammatory conditions, bone loss, and cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows six graphs, each of average arthritic index as a functionof time (dosing day) for test compound dosed at 10 mg/kg/day by oralgavage (open circles) and control (solid circles), for each of: (A)HMC-C-02-A (top left), (B) HMC-C-01-A (top middle), (C) HMC-N-02-A (topright), (D) HMC-N-01-A (bottom left), (E) HMC-C-01-B (bottom middle),(F) HMC-N-01-B (bottom right), as described in Biological Study 6 below.

FIG. 2 shows two graphs, each of arthritic index as a function of time(dosing day) for test compound (open circles, open squares), control(solid circles) and positive control, the marketed drug etanercept(triangles), for each of (A) ABD899 at 10 mg/kg/day (left), (B)HMC-C-01-A at 0.3 mg/kg/day and 3 mg/kg/day (right), as described inBiological Study 6 below.

FIG. 3 shows six graphs, each of average arthritic index as a functionof time (dosing day) for test compound (open circles), control (solidcircles), and positive control, methotrexate (triangles) for each of:(A) ABD899 dosed at 3 mg/kg/day (top left), (B) HMC-C-01-A dosed at 3mg/kg/day (top middle), (C) HMC-N-01-A dosed at 3 mg/kg/day (top right),(D) ABD899 dosed at 10 mg/kg/day (bottom left), (E) HMC-C-01-A dosed at10 mg/kg/day (bottom middle), and (F) HMC-N-01-A dosed at 10 mg/kg/day(bottom right), as described in Biological Study 11 below.

SUMMARY OF THE INVENTION

One aspect of the invention pertains to certain substitutedN-(4-hydroxy-4-methyl-cyclohexyl)-4-phenyl-benzenesulfonamide andN-(4-hydroxy-4-methyl-cyclohexyl)-4-(2-pyridyl)benzenesulfonamidecompounds (collectively referred to herein as HMC compounds), asdescribed herein.

Another aspect of the invention pertains to a composition (e.g., apharmaceutical composition) comprising an HMC compound, as describedherein, and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention pertains to a method of preparing acomposition (e.g., a pharmaceutical composition) comprising the step ofmixing an HMC compound, as described herein, and a pharmaceuticallyacceptable carrier or diluent.

Another aspect of the present invention pertains to an HMC compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy, for example, for use a method of treatment of adisorder (e.g., a disease) as described herein.

Another aspect of the present invention pertains to use of an HMCcompound, as described herein, in the manufacture of a medicament fortreatment, for example, treatment of a disorder (e.g., a disease) asdescribed herein.

Another aspect of the present invention pertains to a method oftreatment, for example, of a disorder (e.g., a disease) as describedherein, comprising administering to a patient in need of treatment atherapeutically effective amount of an HMC compound, as describedherein, preferably in the form of a pharmaceutical composition.

Another aspect of the present invention pertains to a kit comprising (a)an HMC compound, as described herein, preferably provided as apharmaceutical composition and in a suitable container and/or withsuitable packaging; and (b) instructions for use, for example, writteninstructions on how to administer the compound.

Another aspect of the present invention pertains to an HMC compoundobtainable by a method of synthesis as described herein, or a methodcomprising a method of synthesis as described herein.

Another aspect of the present invention pertains to an HMC compoundobtained by a method of synthesis as described herein, or a methodcomprising a method of synthesis as described herein.

Another aspect of the present invention pertains to novel intermediates,as described herein, which are suitable for use in the methods ofsynthesis described herein.

Another aspect of the present invention pertains to the use of suchnovel intermediates, as described herein, in the methods of synthesisdescribed herein.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspects of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Compounds

One aspect of the present invention relates to certain compounds whichmay conveniently be described as substitutedN-(4-hydroxy-4-methyl-cyclohexyl)-4-phenyl-benzenesulfonamide andN-(4-hydroxy-4-methyl-cyclohexyl)-4-(2-pyridyl)benzenesulfonamidecompounds.

Thus, one aspect of the present invention is a compound selected fromcompounds of the following formulae, or a pharmaceutically acceptablesalt, hydrate, or solvate thereof (for convenience, collectivelyreferred to herein as “HMC compounds”):

HMC-C-01

HMC-C-02

HMC-C-03

HMC-C-04

HMC-C-05

HMC-C-06

HMC-N-01

HMC-N-02

HMC-N-03

HMC-N-04

Note that the substituents on one side of the cyclohexyl ring (i.e., —OHand —CH₃ on the right-hand side) may be positioned “trans”/“cis” or“cis”/“trans” with respect to the rest of the molecule (that is, on thecyclohexyl ring to which they attached, with respect to the rest of thecompound which is attached at the para position of the cyclohexyl ring).

“cis-OH”

“cis-OH”

“trans-OH”

“trans-OH”

Unless otherwise indicated, it is intended that all such conformationsare encompassed by a reference to a compound that does not specify aparticular conformation.

In one embodiment, the compound is in the “trans-OH” conformation, asin, for example, the following compounds:

HMC-C-01-A

HMC-C-02-A

HMC-C-03-A

HMC-C-04-A

HMC-C-05-A

HMC-C-06-A

HMC-N-01-A

HMC-N-02-A

HMC-N-03-A

HMC-N-04-A

In one embodiment, the compound is in the “cis-OH” conformation, as in,for example, the following compounds:

HMC-C-01-B

HMC-C-02-B

HMC-C-03-B

HMC-C-04-B

HMC-C-05-B

HMC-C-06-B

HMC-N-01-B

HMC-N-02-B

HMC-N-03-B

HMC-N-04-B

Note also that the cyclohexane ring may take a “chair”, “boat”, or“twist” conformation, and that interconversion between the conformationsis possible. Unless otherwise indicated, it is intended that all suchconformations (e.g., “chair”, “boat”, “twist”, “OH is axial”, “OH isequatorial”, etc.) are encompassed by a reference to a compound thatdoes not specify a particular conformation.

Substantially Purified Forms

One aspect of the present invention pertains to HMC compounds, asdescribed herein, in substantially purified form and/or in a formsubstantially free from contaminants.

In one embodiment, the substantially purified form is at least 50% byweight, e.g., at least 60% by weight, e.g., at least 70% by weight,e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., atleast 95% by weight, e.g., at least 97% by weight, e.g., at least 98% byweight, e.g., at least 99% by weight.

Unless specified, the substantially purified form refers to the compoundin any conformational form. For example, in one embodiment, thesubstantially purified form refers to a mixture of conformational forms,i.e., purified with respect to other compounds. In one embodiment, thesubstantially purified form refers to one conformational form. In oneembodiment, the substantially purified form refers to a mixture ofconformational forms. In one embodiment, the substantially purified formrefers to an equimolar mixture of conformational forms.

In one embodiment, the contaminants represent no more than 50% byweight, e.g., no more than 40% by weight, e.g., no more than 30% byweight, e.g., no more than 20% by weight, e.g., no more than 10% byweight, e.g., no more than 5% by weight, e.g., no more than 3% byweight, e.g., no more than 2% by weight, e.g., no more than 1% byweight.

Unless specified, the contaminants refer to other compounds, that is,other than conformational forms. In one embodiment, the contaminantsrefer to other compounds and other conformational forms.

In one embodiment, the substantially purified form is at least 60%conformationally pure (i.e., 60% of the compound, on a molar basis, isthe desired conformation, and 40% is the undesired conformationalform(s))), e.g., at least 70% conformationally pure, e.g., at least 80%conformationally pure, e.g., at least 90% conformationally pure, e.g.,at least 95% conformationally pure, e.g., at least 97% conformationallypure, e.g., at least 98% conformationally pure, e.g., at least 99%conformationally pure.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diastereoisomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

A reference to a class of structures may well include structurallyisomeric forms falling within that class (e.g., C₁₋₇alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). However,reference to a specific group or substitution pattern is not intended toinclude other structural (or constitutional isomers) which differ withrespect to the connections between atoms rather than by positions inspace. For example, a reference to a methoxy group, —OCH₃, is not to beconstrued as a reference to its structural isomer, a hydroxymethylgroup, —CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to beconstrued as a reference to its structural isomer, meta-chlorophenyl.

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹¹C, ¹²C, ¹³C, and ¹⁴C; O may be in anyisotopic form, including ¹⁵O, ¹⁶O and ¹⁸O; N may be in any isopotic formincluding ¹⁴N and ¹⁵N; F may be in any isopotic form including ¹⁸F and¹⁹F and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including mixtures (e.g., racemicmixtures) thereof. Methods for the preparation (e.g., asymmetricsynthesis) and separation (e.g., fractional crystallisation andchromatographic means) of such isomeric forms are either known in theart or are readily obtained by adapting the methods taught herein, orknown methods, in a known manner.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄+) and substituted ammonium ions (e.g., NH₃R+,NH₂R₂+, NHR₃+, NR₄+). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄+.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃+), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, those derived from the followingorganic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic,ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic,hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic,lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic,oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic,propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric,toluenesulfonic, and valeric. Examples of suitable polymeric organicanions include, but are not limited to, those derived from the followingpolymeric acids: tannic acid, carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular compound alsoincludes salt forms thereof.

Solvates and Hydrates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the compound. The term “solvate” is used hereinin the conventional sense to refer to a complex of solute (e.g.,compound, salt of compound) and solvent. If the solvent is water, thesolvate may be conveniently referred to as a hydrate, for example, amono-hydrate, a di-hydrate, a tri-hydrate, etc.

Unless otherwise specified, a reference to a particular compound alsoincludes solvate and hydrate forms thereof.

Chemically Protected Forms

It may be convenient or desirable to prepare, purify, and/or handle thecompound in a chemically protected form. The term “chemically protectedform” is used herein in the conventional chemical sense and pertains toa compound in which one or more reactive functional groups are protectedfrom undesirable chemical reactions under specified conditions (e.g.,pH, temperature, radiation, solvent, and the like). In practice, wellknown chemical methods are employed to reversibly render unreactive afunctional group, which otherwise would be reactive, under specifiedconditions. In a chemically protected form, one or more reactivefunctional groups are in the form of a protected or protecting group(also known as a masked or masking group or a blocked or blockinggroup). By protecting a reactive functional group, reactions involvingother unprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, Protective Groups inOrganic Synthesis (T. Green and P. Wuts; 4th Edition; John Wiley andSons, 2006).

A wide variety of such “protecting,” “blocking,” or “masking” methodsare widely used and well known in organic synthesis. For example, acompound which has two non-equivalent reactive functional groups, bothof which would be reactive under specified conditions, may bederivatized to render one of the functional groups “protected,” andtherefore unreactive, under the specified conditions; so protected, thecompound may be used as a reactant which has effectively only onereactive functional group. After the desired reaction (involving theother functional group) is complete, the protected group may be“deprotected” to return it to its original functionality.

For example, an amine group may be protected, for example, as an amide(—NRCO—R) or a urethane (—NRCO—OR), for example, as: a methyl amide(—NHCO—CH₃); a benzyloxy amide (—NHCO—OCH₂C₆H₅, —NH-Cbz); as a t-butoxyamide (—NHCO—OC(CH₃)₃, —NH-Boc); a 2-biphenyl-2-propoxy amide(—NHCO—OC(CH₃)₂C₆H₄C₆H₅, —NH-Bpoc), as a 9-fluorenylmethoxy amide(—NH—Fmoc), as a 6-nitroveratryloxy amide (—NH—Nvoc), as a2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxyamide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a2(-phenylsulfonyl)ethyloxy amide (—NH—Psec); or, in suitable cases(e.g., cyclic amines), as a nitroxide radical (>N—O•).

Prodrugs

It may be convenient or desirable to prepare, purify, and/or handle thecompound in the form of a prodrug. The term “prodrug,” as used herein,pertains to a compound which, when metabolised (e.g., in vivo), yieldsthe desired active compound. Typically, the prodrug is inactive, or lessactive than the desired active compound, but may provide advantageoushandling, administration, or metabolic properties.

Chemical Synthesis

Methods for the chemical synthesis of HMC compounds are describedherein. These and/or other well-known methods may be modified and/oradapted in known ways in order to facilitate the synthesis of additionalHMC compounds described herein.

Compositions

One aspect of the present invention pertains to a composition (e.g., apharmaceutical composition) comprising an HMC compound, as describedherein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

In one embodiment, the composition further comprises one or more (e.g.,1, 2, 3, 4) additional therapeutic agents, as described herein.

Another aspect of the present invention pertains to a method ofpreparing a composition (e.g., a pharmaceutical composition) comprisingadmixing an HMC compound, as described herein, and a pharmaceuticallyacceptable carrier, diluent, or excipient.

Another aspect of the present invention pertains to a method ofpreparing a composition (e.g., a pharmaceutical composition) comprisingadmixing an HMC compound, as described herein; one or more (e.g., 1, 2,3, 4) additional therapeutic agents, as described herein; and apharmaceutically acceptable carrier, diluent, or excipient.

Uses

The HMC compounds, as described herein, are useful, for example, in thetreatment of disorders (e.g., diseases) including, for example, thedisorders (e.g., diseases) described herein.

Use in Methods of Therapy

Another aspect of the present invention pertains to an HMC compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy, for example, for use a method of treatment of adisorder (e.g., a disease) as described herein.

Another aspect of the present invention pertains to an HMC compound, asdescribed herein, in combination with one or more (e.g., 1, 2, 3, 4)additional therapeutic agents, as described herein, for use in a methodof treatment of the human or animal body by therapy, for example, foruse a method of treatment of a disorder (e.g., a disease) as describedherein.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of an HMCcompound, as described herein, in the manufacture of a medicament fortreatment, for example, treatment of a disorder (e.g., a disease) asdescribed herein.

In one embodiment, the medicament comprises the HMC compound.

Another aspect of the present invention pertains to use of an HMCcompound, as described herein, and one or more (e.g., 1, 2, 3, 4)additional therapeutic agents, as described herein, in the manufactureof a medicament for treatment, for example, treatment of a disorder(e.g., a disease) as described herein.

In one embodiment, the medicament comprises the HMC compound and the oneor more (e.g., 1, 2, 3, 4) additional therapeutic agents.

Methods of Treatment

Another aspect of the present invention pertains to a method oftreatment, for example, of a disorder (e.g., a disease) as describedherein, comprising administering to a patient in need of treatment atherapeutically effective amount of an HMC compound, as describedherein, preferably in the form of a pharmaceutical composition.

Another aspect of the present invention pertains to a method oftreatment, for example, of a disorder (e.g., a disease) as describedherein, comprising administering to a patient in need of treatment atherapeutically effective amount of an HMC compound, as describedherein, preferably in the form of a pharmaceutical composition, and oneor more (e.g., 1, 2, 3, 4) additional therapeutic agents, as describedherein, preferably in the form of a pharmaceutical composition.

Conditions Treated

In one embodiment, the treatment is treatment of an inflammatorydisorder or an autoimmune disorder.

In one embodiment, the treatment is treatment of a disorder associatedwith inflammation and/or activation of the immune system.

In one embodiment, the treatment is treatment of a disorder mediated byexcessive and/or inappropriate and/or prolonged activation of the immunesystem.

In one embodiment, the treatment is treatment of inflammation.

In one embodiment, the treatment is treatment of a disorder associatedwith inflammation or activation of the immune system.

In one embodiment, the treatment is treatment of rheumatoid arthritis;psoriasis; psoriatic arthritis; chronic obstructive pulmonary disease(COPD); asthma; atherosclerosis; inflammatory bowel disease; orankylosing spondylitis.

In one embodiment, the treatment is treatment of rheumatoid arthritis.

In one embodiment, the treatment is treatment of psoriasis.

In one embodiment, the treatment is treatment of psoriatic arthritis.

In one embodiment, the treatment is treatment of chronic obstructivepulmonary disease (COPD).

In one embodiment, the treatment is treatment of asthma.

In one embodiment, the treatment is treatment of atherosclerosis.

In one embodiment, the treatment is treatment of ankylosing spondylitis.

In one embodiment, the treatment is treatment of inflammatory boweldisease.

In one embodiment, the treatment is prevention of an immune responseleading to organ or graft rejection following transplant.

In one embodiment, the treatment is prevention of an inflammatorycondition in which IRF-5 expression or activity is aberrant.

In one embodiment, the treatment is treatment of a tumour which overexpresses TNFα, IL-1, IL-6, RANKL, and/or NFκB.

In one embodiment, the treatment is treatment of a tumour for whichinhibition of TNFα, IL-1, RANKL, NFκB, IRFs such as IRF-3, -5 or -7and/or IL-6 expression or activity or signalling facilitates or improvesthe action of cytotoxic tumouricidal agents.

In one embodiment, the treatment is treatment of a haematologicalmalignancy.

In one embodiment, the treatment is treatment of multiple myeloma.

In one embodiment, the treatment is treatment of leukaemia; e.g., acutelymphoblastic leukaemia.

In one embodiment, the treatment is treatment of lymphoma; e.g.,non-Hodgkin's Lymphoma, T-cell lymphoma (e.g., T-lymphoblastic lymphoma,extranodal T-cell lymphoma, cutaneous T-cell lymphoma, anaplastic largecell lymphoma, angioimmunoblastic T-cell lymphoma), and B-cell lymphoma(e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma) (e.g., diffuse largeB-cell lymphoma, follicular lymphoma, mucosa-associated lymphatic tissuelymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, hairycell leukaemia and Burkitt's lymphoma).

In one embodiment, the treatment is treatment of a solid tumour cancer,e.g., bladder cancer, breast cancer (female and/or male), colon cancer,renal cell carcinoma, kidney cancer, lung cancer, pancreatic cancer,gastric cancer, prostate cancer, brain cancer, skin cancer, thyroidcancer, basal cell ameloblastoma, or melanoma.

In one embodiment, the haematological malignancy (e.g., multiplemyeloma, leukaemia, lymphoma, etc.) and the solid tumour cancer (e.g.,cancer of the bladder, etc.) is associated with activation of NFκB, withaberrant NFκB signalling, or with inflammation.

In one embodiment, the haematological malignancy (e.g., multiplemyeloma, leukaemia, lymphoma, etc.) and the solid tumour cancer (e.g.,cancer of the bladder, etc.) is associated with inactivation orimpairment of caspase induction or with aberrant caspase signalling.

In one embodiment, the treatment is treatment of a proliferativedisorder; e.g., Castleman's disease.

In one embodiment, the treatment is treatment of a disease or disorderselected from: diseases having an inflammatory or autoimmune component,including asthma, atherosclerosis, allergic diseases, such as atopy,allergic rhinitis, atopic dermatitis, anaphylaxis, allergicbronchopulmonary aspergillosis, and hypersensitivity pneumonitis (pigeonbreeders disease, farmer's lung disease, humidifier lung disease, maltworkers' lung disease); allergies, including flea allergy dermatitis inmammals such as domestic animals, e.g., dogs and cats, contact allergensincluding mosquito bites or other insect sting allergies, poison ivy,poison oak, poison sumac, or other skin allergens; autoimmune disorders,including type I diabetes and associated complications, multiplesclerosis, arthritis, systemic lupus erythematosus, autoimmune(Hasimoto's) thyroiditis, autoimmune liver diseases such as hepatitisand primary biliary cirrhosis, hyperthyroidism (Graves' disease;thyrotoxicosis), insulin-resistant diabetes, autoimmune adrenalinsufficiency (Addison's disease), autoimmune oophoritis, autoimmuneorchitis, autoimmune haemolytic anaemia, paroxysmal cold hemoglobinuria,Behçet's disease, autoimmune thrombocytopenia, autoimmune neutropenia,pernicious anaemia, pure red cell anaemia, autoimmune coagulopathies,endometriosis, myasthenia gravis, experimental allergicencephalomyelitis, autoimmune polyneuritis, pemphigus and other bullousdiseases, rheumatic carditis, Goodpasture's syndrome, postcardiotomysyndrome, Sjogren's syndrome, polymyositis, dermatomyositis, andscleroderma; disease states resulting from inappropriate inflammation,either local or systemic, for example, irritable or inflammatory bowelsyndrome (Mazzucchelli et al., 1996), skin diseases such as lichenplanus, delayed type hypersensitivity, chronic pulmonary inflammation,e.g., pulmonary alveolitis and pulmonary granuloma, gingivalinflammation or other periodontal disease, and osseous inflammationassociated with lesions of endodontic origin (Volejnikova et al., 1997),hypersensitivity lung diseases such as hypersensitivity pneumonitis(Sugiyama et al., 1995), and inflammation related to histamine releasefrom basophils (Dvorak et al., 1996), such as hay fever, histaminerelease from mast cells (Galli et al., 1989), or mast cell tumours,types of type 1 hypersensitivity reactions (anaphylaxis, skin allergy,hives, gout, allergic rhinitis, and allergic gastroenteritis);ulcerative colitis or Crohn's disease; TNFα induced polycystic kidneydisease (Li et al., 2008); or Cryopyrin-Associated Periodic Syndromes,including Muckle-Wells Syndrome.

In one embodiment, the treatment is treatment of a disorder mediated byosteoclasts.

In one embodiment, the treatment is treatment of a disordercharacterised by excessive bone resorption.

In one embodiment, the treatment is treatment of a disorder associatedwith bone loss.

In one embodiment, the treatment is treatment of bone loss.

In one embodiment, the treatment is treatment of bone loss associatedwith inflammation.

In one embodiment, the treatment is treatment of bone loss notassociated with inflammation.

In one embodiment, the treatment is treatment of bone loss associatedwith excessive osteoclast activation.

In one embodiment, the treatment is treatment of joint destruction.

In one embodiment, the treatment is treatment of joint destructionassociated with inflammation.

In one embodiment, the treatment is treatment of joint destructionassociated with excessive osteoclast activation.

In one embodiment, the treatment is treatment of bone loss associatedwith excessive osteoclast activation in rheumatoid arthritis,osteoporosis, cancer-associated bone disease, or Paget's disease.

In one embodiment, the treatment is treatment of bone loss associatedwith rheumatoid arthritis, osteoporosis, cancer-associated bone disease,or Paget's disease of bone.

In one embodiment, the treatment is treatment of rheumatoid arthritis,osteoporosis, cancer-associated bone disease, or Paget's disease ofbone.

In one embodiment, the treatment is treatment of neoplasia of bones,whether as a primary tumour or as metastases, including osteosarcoma andosteoma (see, e.g., Zheng et al., 1998) and cancer-associated bonedisease (e.g., hypercalcaemia of malignancy, bone metastases, osteolyticbone metastases, multiple myeloma, breast carcinoma).

In one embodiment, the treatment is treatment of hypercalcaemia causedby conditions associated with increased bone resorption, including:vitamin D intoxication, primary or tertiary hyperparathyroidism,immobilisation, and sarcoidosis.

In one embodiment, the treatment is treatment of aseptic loosening ofprosthetic implants (e.g., artificial joints, e.g., knees, hips, etc.,can loosen due to osteoclast activity driven by local inflammation)(see, e.g., Childs et al., 2001).

In one embodiment, the treatment is treatment of osteopetrosis,osteoarthritis, or ectopic bone formation.

In one embodiment, the treatment is treatment of a disorder associatedwith fibrosis, such as systemic sclerosis or scleroderma.

In one embodiment, the treatment is treatment of a rare vasculitide,such as Behçet's disease.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, alleviation of symptoms of thecondition, amelioration of the condition, and cure of the condition.Treatment as a prophylactic measure (i.e., prophylaxis) is alsoincluded. For example, use with patients who have not yet developed thecondition, but who are at risk of developing the condition, isencompassed by the term “treatment.”

For example, treatment of inflammation includes the prophylaxis ofinflammation, reducing the incidence of inflammation, reducing theseverity of inflammation, alleviating the symptoms of inflammation, etc.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound, or a material, composition or dosage formcomprising a compound, which is effective for producing some desiredtherapeutic effect, commensurate with a reasonable benefit/risk ratio,when administered in accordance with a desired treatment regimen.

Combination Therapies

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. For example, the compounds describedherein may also be used in combination therapies, e.g., in conjunctionwith other agents, for example, anti-inflammation agents, etc. Examplesof treatments and therapies include chemotherapy (the administration ofactive agents, including, e.g., drugs, antibodies (e.g., as inimmunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT,ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; genetherapy; and controlled diets.

One aspect of the present invention pertains to a compound as describedherein, in combination with one or more additional therapeutic agents.

The particular combination would be at the discretion of the physicianwho would select dosages using his common general knowledge and dosingregimens known to a skilled practitioner.

The agents (i.e., the compound described herein, plus one or more otheragents) may be administered simultaneously or sequentially, and may beadministered in individually varying dose schedules and via differentroutes. For example, when administered sequentially, the agents can beadministered at closely spaced intervals (e.g., over a period of 5-10minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart,or even longer periods apart where required), the precise dosage regimenbeing commensurate with the properties of the therapeutic agent(s).

The agents (i.e., the compound described here, plus one or more otheragents) may be formulated together in a single dosage form, oralternatively, the individual agents may be formulated separately andpresented together in the form of a kit, optionally with instructionsfor their use.

Other Uses

The HMC compounds described herein may also be used as part of an invitro assay, for example, in order to determine whether a candidate hostis likely to benefit from treatment with the compound in question.

The HMC compounds described herein may also be used as a standard, forexample, in an assay, in order to identify other compounds, otheranti-inflammation agents, etc.

Kits

One aspect of the invention pertains to a kit comprising (a) an HMCcompound as described herein, or a composition comprising an HMCcompound as described herein, e.g., preferably provided in a suitablecontainer and/or with suitable packaging; and (b) instructions for use,e.g., written instructions on how to administer the compound orcomposition.

In one embodiment, the kit further comprises one or more (e.g., 1, 2, 3,4) additional therapeutic agents, as described herein.

The written instructions may also include a list of indications forwhich the active ingredient is a suitable treatment.

Routes of Administration

The HMC compound or pharmaceutical composition comprising the HMCcompound may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or topically (i.e., atthe site of desired action).

Routes of administration include oral (e.g., by ingestion); buccal;sublingual; transdermal (including, e.g., by a patch, plaster, etc.);transmucosal (including, e.g., by a patch, plaster, etc.); intranasal(e.g., by nasal spray, drops or from an atomiser or dry powder deliverydevice); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation orinsufflation therapy using, e.g., an aerosol, e.g., through the mouth ornose); rectal (e.g., by suppository or enema); vaginal (e.g., bypessary); parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, and intrasternal; by implant of a depot or reservoir, forexample, subcutaneously or intramuscularly.

In one preferred embodiment, the route of administration is oral (e.g.,by ingestion).

In one preferred embodiment, the route of administration is parenteral(e.g., by injection).

The Subject/Patient

The subject/patient may be a chordate, a vertebrate, a mammal, aplacental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g.,a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), alagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog),feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig),ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., amonkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g.,gorilla, chimpanzee, orangutang, gibbon), or a human. Furthermore, thesubject/patient may be any of its forms of development, for example, afoetus.

In one preferred embodiment, the subject/patient is a human.

Formulations

While it is possible for the HMC compound to be administered alone, itis preferable to present it as a pharmaceutical formulation (e.g.,composition, preparation, medicament) comprising at least one HMCcompound, as described herein, together with one or more otherpharmaceutically acceptable ingredients well known to those skilled inthe art, including pharmaceutically acceptable carriers, diluents,excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants,lubricants, stabilisers, solubilisers, surfactants (e.g., wettingagents), masking agents, colouring agents, flavouring agents, andsweetening agents. The formulation may further comprise other activeagents, for example, other therapeutic or prophylactic agents.

Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one HMC compound, as describedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, e.g., carriers,diluents, excipients, etc. If formulated as discrete units (e.g.,tablets, etc.), each unit contains a predetermined amount (dosage) ofthe compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbookof Pharmaceutical Excipients, 5th edition, 2005.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association thecompound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the compound with carriers (e.g.,liquid carriers, finely divided solid carrier, etc.), and then shapingthe product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations may suitably be in the form of liquids, solutions (e.g.,aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous),emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups,electuaries, mouthwashes, drops, tablets (including, e.g., coatedtablets), granules, powders, lozenges, pastilles, capsules (including,e.g., hard and soft gelatin capsules), cachets, pills, ampoules,boluses, suppositories, pessaries, tinctures, gels, pastes, ointments,creams, lotions, oils, foams, sprays, mists, or aerosols.

Formulations may suitably be provided as a patch, adhesive plaster,bandage, dressing, or the like which is impregnated with one or morecompounds and optionally one or more other pharmaceutically acceptableingredients, including, for example, penetration, permeation, andabsorption enhancers. Formulations may also suitably be provided in theform of a depot or reservoir.

The compound may be dissolved in, suspended in, or admixed with one ormore other pharmaceutically acceptable ingredients. The compound may bepresented in a liposome or other microparticulate which is designed totarget the compound, for example, to blood components or one or moreorgans.

Formulations suitable for oral administration (e.g., by ingestion)include liquids, solutions (e.g., aqueous, non-aqueous), suspensions(e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water,water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders,capsules, cachets, pills, ampoules, boluses.

Formulations suitable for buccal administration include mouthwashes,lozenges, pastilles, as well as patches, adhesive plasters, depots, andreservoirs. Lozenges typically comprise the compound in a flavoredbasis, usually sucrose and acacia or tragacanth. Pastilles typicallycomprise the compound in an inert matrix, such as gelatin and glycerin,or sucrose and acacia. Mouthwashes typically comprise the compound in asuitable liquid carrier.

Formulations suitable for sublingual administration include tablets,lozenges, pastilles, capsules, and pills.

Formulations suitable for oral transmucosal administration includeliquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g.,aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil),mouthwashes, lozenges, pastilles, as well as patches, adhesive plasters,depots, and reservoirs.

Formulations suitable for non-oral transmucosal administration includeliquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g.,aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil),suppositories, pessaries, gels, pastes, ointments, creams, lotions,oils, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for transdermal administration include gels,pastes, ointments, creams, lotions, and oils, as well as patches,adhesive plasters, bandages, dressings, depots, and reservoirs.

Tablets may be made by conventional means, e.g., compression ormoulding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine thecompound in a free-flowing form such as a powder or granules, optionallymixed with one or more binders (e.g., povidone, gelatin, acacia,sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g., lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, silica);disintegrants (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g., sodium lauryl sulfate);preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid); flavours, flavour enhancing agents, and sweeteners.Moulded tablets may be made by moulding in a suitable machine a mixtureof the powdered compound moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the compound therein using, forexample, hydroxypropylmethyl cellulose in varying proportions to providethe desired release profile. Tablets may optionally be provided with acoating, for example, to affect release, for example an enteric coating,to provide release in parts of the gut other than the stomach.

Ointments are typically prepared from the compound and a paraffinic or awater-miscible ointment base.

Creams are typically prepared from the compound and an oil-in-watercream base. If desired, the aqueous phase of the cream base may include,for example, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycoland mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the compoundthrough the skin or other affected areas. Examples of such dermalpenetration enhancers include dimethylsulfoxide and related analogues.

Emulsions are typically prepared from the compound and an oily phase,which may optionally comprise merely an emulsifier (otherwise known asan emulgent), or it may comprises a mixture of at least one emulsifierwith a fat or an oil or with both a fat and an oil. Preferably, ahydrophilic emulsifier is included together with a lipophilic emulsifierwhich acts as a stabiliser. It is also preferred to include both an oiland a fat. Together, the emulsifier(s) with or without stabiliser(s)make up the so-called emulsifying wax, and the wax together with the oiland/or fat make up the so-called emulsifying ointment base which formsthe oily dispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulfate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required. Alternatively, high melting point lipids suchas white soft paraffin and/or liquid paraffin or other mineral oils canbe used.

Formulations suitable for intranasal administration, where the carrieris a liquid, include, for example, nasal spray, nasal drops, or byaerosol administration by nebuliser, include aqueous or oily solutionsof the compound.

Formulations suitable for intranasal administration, where the carrieris a solid, include, for example, those presented as a coarse powderhaving a particle size, for example, in the range of about 20 to about500 microns which is administered in the manner in which snuff is taken,i.e., by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose.

Formulations suitable for pulmonary administration (e.g., by inhalationor insufflation therapy) include those presented as an aerosol sprayfrom a pressurised pack, with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for ocular administration include eye dropswherein the compound is dissolved or suspended in a suitable carrier,especially an aqueous solvent for the compound.

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, natural orhardened oils, waxes, fats, semi-liquid or liquid polyols, for example,cocoa butter or a salicylate; or as a solution or suspension fortreatment by enema.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the compound, such carriers as are known inthe art to be appropriate.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the compound isdissolved, suspended, or otherwise provided (e.g., in a liposome orother microparticulate). Such liquids may additional contain otherpharmaceutically acceptable ingredients, such as anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, suspending agents, thickeningagents, and solutes which render the formulation isotonic with the blood(or other relevant bodily fluid) of the intended recipient. Examples ofexcipients include, for example, water, alcohols, polyols, glycerol,vegetable oils, and the like. Examples of suitable isotonic carriers foruse in such formulations include Sodium Chloride Injection, Ringer'sSolution, or Lactated Ringer's Injection. Typically, the concentrationof the compound in the liquid is from about 1 ng/mL to about 10 μg/mL,for example, from about 10 ng/mL to about 1 μg/mL. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the HMC compounds, and compositions comprising the HMCcompounds, can vary from patient to patient. Determining the optimaldosage will generally involve the balancing of the level of therapeuticbenefit against any risk or deleterious side effects. The selecteddosage level will depend on a variety of factors including the activityof the particular HMC compound, the route of administration, the time ofadministration, the rate of excretion of the HMC compound, the durationof the treatment, other drugs, compounds, and/or materials used incombination, the severity of the condition, and the species, sex, age,weight, condition, general health, and prior medical history of thepatient. The amount of HMC compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the HMC compound is in the range of about50 μg to about 20 mg (more typically about 100 μg to about 10 mg) perkilogram body weight of the subject per day. For pulmonaryadministration (e.g., by inhalation), a suitable dosage is in the rangeof about 50 ng to about 1 mg per kilogram body weight of the subject perday. Where the compound is a salt, an ester, an amide, a prodrug, or thelike, the amount administered is calculated on the basis of the parentcompound and so the actual weight to be used is increasedproportionately.

Chemical Synthesis

Methods for the chemical synthesis of the HMC compounds are describedherein. These and/or other well-known methods (see, e.g., Greig et al.,2010a; Bahmanyar et al., 2010) may be modified and/or adapted in knownways in order to provide alternative or improved methods of synthesis.

Palladium hydroxide (50% wet with water; 2.0 g) was added to a stirredsolution of (1r,4r)-4-(dibenzylamino)-1-methylcyclohexanol (7.5 g, 24.2mmol) in methanol (100 mL) in a 300 mL autoclave. The autoclave wascharged with hydrogen (50 atm; ˜5 MPa) and heated at 80° C. for 24hours. The mixture was cooled and the catalyst filtered off. Thefiltrate was returned to the autoclave and palladium hydroxide (50% wetwith water; 3.0 g) was added. The autoclave was charged with hydrogen(50 atm; ˜50 MPa) and heated at 80° C. overnight. The mixture was cooledand filtered through celite and the filtrate was concentrated to givethe title compound as an off-white gummy solid (3.2 g, quant.).

¹H NMR: (400 MHz; CDCl₃) δ 2.86-2.76 (1H, m), 1.84-1.76 (2H, m),1.75-1.63 (2H, m), 1.55-1.43 (2H, m), 1.30-1.17 (5H, m).

Four equal batches of (1s,4s)-4-dibenzylamino-1-methylcyclohexan-1-ol(each batch was 15 g, total 60 g) were separately debenzylated asfollows: To (1s,4s)-4-dibenzylamino-1-methylcyclohexan-1-ol (15 g, 193.9mmol) in ethanol (450 mL) was added 10% palladium hydroxide (15 g, 50%wet catalyst). The reaction mixtures were flushed with nitrogen followedby hydrogen gas and stirred under an atmosphere of hydrogen for 16 hoursat room temperature. The solution was filtered through celite and whichwas washed with additional ethyl acetate. The filtrates from all fourbatches were combined and evaporated under reduced pressure to affordthe title compound (23 g, 91.8% yield). The compound was used withoutfurther purification in the next step.

¹H NMR (400 MHz, CDCl₃) δ: 2.6 (m, 1H), 1.74-1.56 (m, 4H), 1.5-1.3 (m,7H), 1.21 (s, 3H).

Diisopropylethylamine (24 mL, 137.8 mmol) was added to a solution of(1r,4r)-4-amino-1-methylcyclohexanol (3.6 g, 27.86 mmol) indichloromethane (150 mL) and the reaction mixture was cooled to 0° C.4-Bromobenzene-1-sulfonyl chloride (7.83 g, 30.6 mmol) was added assolid and the reaction mixture was allowed to stir at room temperaturefor 4 hours. The reaction mixture was neutralized with 1 M hydrochloricacid and the compound was extracted into dichloromethane. The organiclayer was separated, dried over sodium sulfate and concentrated underreduced pressure. The residue obtained was washed with pentane, filteredand dried to give the title compound (7 g, 72%).

¹H NMR: (400 MHz; CDCl₃) δ 7.74 (2H, d), 7.65 (2H, d), 4.77-4.61 (1H,m), 3.33-3.23 (1H, m), 1.85-1.75 (2H, m), 1.63-1.51 (2H, m), 1.49-1.30(4H, m), 1.20 (3H, s).

LCMS: (Run time: 3.5 min): Retention time: 1.33 min (97%, MS (ESI) m/z346 (M−H)⁺).

A solution of4-bromo-N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)benzenesulfonamide (9 g,25.8 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane)(9.87 g, 38.9 mmol) and potassium acetate (7.6 g, 77.5 mmol), in toluene(50 mL) was degassed using argon for 10 min. [1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.8 g, 2.5 mmol) was added and thereaction mixture was degassed for another 10 min and stirred at 100° C.for 4 hours. The solvent was evaporated under reduced pressure and thecompound was extracted into ethyl acetate. The organic layer wasseparated, dried over sodium sulfate and concentrated under reducedpressure to give the title compound (8 g, 78%) MS (ESI) m/z 394 (M−H)⁺).For large scale batches, the compound was used without furtherpurification. When this preparation was performed on a smaller scale,the residue was taken up in ether, filtered and the filtrate wasconcentrated to give the desired product.

A mixture of dioxane and water (3:1; 20 mL) was degassed.2,3,5-Trichloropyridine (1.65 g, 9.0 mmol),N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(1.8 g, 4.6 mmol), K₂CO₃ (1.24 g, 9.0 mmol) and[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (257 mg, 0.35mmol) were added and the reaction mixture stirred at 120° C. in amicrowave oven for 3 hours. The reaction mixture was concentrated,diluted with ethyl acetate and washed with water. The organic extractswere dried (MgSO₄) and concentrated to give a crude residue which waspurified by flash column (eluent: 40 to 50% ethyl acetate in heptane).The product was purified further by crystallisation three times (ethylacetate/heptane) to give the title compound (284 mg, 15%).

¹H NMR: (400 MHz; CDCl₃) δ 8.59 (1H, m), 7.97 (2H, d), 7.92-7.85 (3H,m), 4.62-4.55 (1H, m), 3.38-3.28 (1H, m), 1.92-1.76 (2H, m), 1.8-1.35(7H, m), 1.22 (3H, s).

LCMS: mobile phase A: 0.05% trifluoroacetic acid in water, mobile phaseB: 0.05% trifluoroacetic acid in acetonitrile; Column: YMC ODS A, C18(50×4.6 mm) 3 uM; Flow rate: 1.2 mL/min; Temperature: Ambient. Run time:4.5 min—starting solvent 20:80 B:A is increased linearly to 95:5 B:Aover the first 3 min, held at 95:5 B:A for 0.5 min then immediatelyreturned to 20:80 B:A for the last 1.5 min. Retention time: 2.50 min,m/z 415 (M+H)⁺.

A stirred solution of dioxane: water (9:1; 100 mL),N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(20 g, 50.6 mmol), 2-bromo-3,5-difluoropyridine (14.73 g, 75.94 mmol)and sodium carbonate (10.73 g, 101.2 mmol) was degassed using argon for10 minutes. [1,1-Bis(diphenylphosphino) ferrocene] dichloropalladium(II)(3.7 g, 5.06 mmol) was added and the reaction mixture was degassed foranother 10 minutes and stirred at 110° C. for 6 hours. The solvent wasevaporated under reduced pressure and the compound was extracted intoethyl acetate. The organic layer was separated, dried over sodiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography and the fractions wereconcentrated to minimum volume and then filtered. The residue obtainedwas washed with 20% ethyl acetate in hexane followed by n-pentane togive the title compound (8 g, 41%).

¹H NMR: (400 MHz; methanol-d₄) δ 8.54 (d, J=2.0 Hz, 1H), 8.11 (d, J=8.2Hz, 2H), 7.99 (d, J=8.1 Hz, 2H), 7.74 (m, 1H), 3.20 (m, 1H), 1.83-1.27(m, 8H), 1.18 (s, 3H).

LCMS: mobile phase A: 10 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 1.75 mins, held at 95:5 B:A for 1 min, reduced linearly to30:70 B:A over 1.25 min and held at 30:70 B:A for the final 0.5 min.Retention time 1.88 min, m/z 381 (M−H)⁺.

A stirred solution of dioxane: water (9:1; 100 mL),N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(12 g, 30.4 mmol), 2-bromo-5-chlorobenzonitrile (9.86 g, 45.5 mmol) andsodium carbonate (6.44 g, 60.8 mmol) was degassed using argon for 10minutes. [1,1-Bis(diphenylphosphino)ferrocene] dichloropalladium(II)(2.21 g, 3.0 mmol) was added and the reaction mixture was degassed foranother 10 minutes and stirred at 110° C. for 6 hours. The solvent wasevaporated under reduced pressure and the compound was extracted intoethyl acetate. The organic layer was separated, dried over sodiumsulfate and concentrated under reduced pressure followed by washing withn-pentane (twice) and dried under vacuum at 45-50° C. to give the titlecompound (5.7 g, 46.5%).

¹H NMR (400 MHz, methanol-d₄) δ 8.01 (d, J=8.1 Hz, 2H), 7.95 (m, 1H),7.83-7.74 (m, 3H), 7.64 (d, J=8.5 Hz, 1H), 3.25-3.17 (m, 1H), 1.8-1.54(m, 4H), 1.47-1.34 (m, 4H), 1.18 (s, 3H).

LCMS: mobile phase A: 10 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2.5 mins, held at 95:5 B:A for 0.5 min, reduced linearly to30:70 B:A over 1 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.167 min, m/z 403 (M−H)⁺.

To a 10 L flask was charged finely divided(1r,4r)-4-amino-1-methylcyclohexan-1-ol (123.7 g, 0.957 mol) anddichloromethane (2400 mL). Triethylamine (534 mL, 3.830 mol) was addeddropwise. The suspension cooled to below 5° C. and4-(4-chloro-2-cyanophenyl)benzene-1-sulfonyl chloride (298.9 g, 0.957mol) in dichloromethane (768 mL) added dropwise maintaining thetemperature at less than 25° C. The reaction mixture was allowed to warmto room temperature and stirred for 40 hours. The reaction mixture wascooled to below 10° C. and 2 M aqueous hydrochloric acid (2090 mL) addeddropwise while maintaining the temperature at less than 25° C.(exothermic addition and white fumes observed). The phases wereseparated and the organic layer was washed with water (2090 mL). Theorganics were dried over anhydrous magnesium sulphate, filtered, and theresidue was washed with dichloromethane (2×50 mL). The combinedfiltrates were then combined with the crude product from a similarsmaller scale reaction of 4-(4-chloro-2-cyanophenyl)benzene-1-sulfonylchloride (50 g) and (1r,4r)-4-amino-1-methylcyclohexan-1-ol, and all ofthe combined materials adsorbed directly onto silica (800 g). This waspurified by chromatography on silica (8000 g) eluting initially withethyl acetate: dichloromethane 20:80, and then sequentially withmixtures of ethyl acetate: dichloromethane 30:70, 40:60, 50:50, followedby neat ethyl acetate. Fractions containing the product were combinedand concentrated to afford a yellow solid. This material was dried in avacuum oven overnight at 40° C. to afford the title compound (406.8 g;overall 88% yield). Analysis by NMR indicated a purity of >97%.

¹H NMR: (270 MHz; CDCl₃) δ 8.02 (d, J=8.6 Hz, 2H), 7.78 (d, J=2 Hz, 1H),7.73-7.63 (m, 3H), 7.49 (d, J=8.5 Hz, 1H), 4.89 (d, J=7 Hz, 1H), 3.38(m, 1H), 1.98-1.75 (m, 2H), 1.75-1.3 (m, 7H, m), 1.23 (s, 3H).

HPLC: mobile phase A: purified water+0.1% trifluoroacetic acid, mobilephase B: acetonitrile+0.1% trifluoroacetic acid; Column: Fortis C184.6×150 mm; 3 uM; Flow rate: 1.0 mL/min. Run time: 30 mins—startingsolvent 5:95 B:A is increased linearly to 95:5 B:A over the first 15mins, held at 95:5 B:A for the final 15 min. Retention time 12.0 min.Mass Spectrum: Bruker Esquire 3000 Plus Ion Trap MS; Positive ionpolarity, ESI: m/z 403 (M−H)⁺.

A stirred solution of dioxane: water (9:1; 100 mL),N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide-yl)benzenesulfonamide(20 g, 50.6 mmol), 1-bromo-4-fluoro-2-(trifluoromethyl)benzene (18.45 g,75.9 mmol) and sodium carbonate (10.73 g, 101.2 mmol) was degassed usingargon for 10 minutes.[1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (3.7 g, 5.06mmol) was added and the reaction mixture was degassed for another 10minutes and stirred at 110° C. for 6 hours. The solvent was evaporatedunder reduced pressure and the compound was extracted into ethylacetate. The organic layer was separated, dried over sodium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography and the fractions were concentrated and thenfiltered. The residue obtained was washed with a minimal volume ofdichloromethane followed by washing with n-pentane (twice) and driedunder vacuum at 45-50° C. to give the title compound (10.2 g, 47%).

¹H NMR (400 MHz, methanol-d₄) δ 7.93 (d, J=7.9 Hz, 2H), 7.59 (d, J=9.2Hz, 1H), 7.51 (d, J=8.1 Hz, 2H), 7.46 (d, J=6.6 Hz, 2H), 3.20 (m, 1H),1.79-1.51 (m, 4H), 1.49-1.30 (m, 4H), 1.18 (s, 3H).

LCMS: mobile phase A: 10 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 1.75 mins, held at 95:5 B:A for 1 min, reduced linearly to30:70 B:A over 1.25 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.16 min, m/z 430 (M−H)⁺.

A mixture of dioxane: water (3:1; 20 mL) was degassed.1-Bromo-2,4,6-trifluorobenzene (1.91 g, 9.1 mmol),N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(1.8 g, 4.6 mmol), K₂CO₃ (1.24 g, 9.0 mmol) and [1,1Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (257 mg, 0.35mmol) were added and the reaction mixture stirred at 120° C. in amicrowave oven for 3 hours. The reaction mixture was concentrated,diluted with ethyl acetate and washed with water. The organic extractswere dried (MgSO₄) and concentrated to give a crude residue which waspurified by flash column (eluent: 40 to 50% ethyl acetate in heptane).The product was purified further by crystallisation (ethylacetate/heptane) to give the title compound (620 mg, 34%).

¹H NMR: (400 MHz; CDCl₃) δ 7.95 (d, 2H), 7.58 (d, 2H), 6.80 (t, 2H),4.62-4.52 (m, 1H), 3.41-3.30 (m, 1H), 1.91-1.81 (m, 2H), 1.66-1.34 (m,6H), 1.22 (s, 3H).

LCMS: mobile phase A: 10 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 5.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2.5 mins, held at 95:5 B:A for 1 min, reduced linearly to30:70 B:A over 1.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.40 min m/z 400 (M+H)⁺.

Diisopropylethylamine (20 mL, 116.2 mmol) was added to a solution of(1r,4r)-4-amino-1-methylcyclohexanol (3 g, 23.2 mmol) in dichloromethane(150 mL) and the reaction mixture was cooled to 0° C.4-Bromo-3-fluorobenzene-1-sulfonyl chloride (6.98 g, 25.5 mmol) wasadded as solid and the reaction mixture was allowed to stir at roomtemperature for 4 hours. The reaction mixture was neutralized with 1 Mhydrochloric acid and the compound was extracted into dichloromethane.The organic layer was separated, dried over sodium sulfate andconcentrated under reduced pressure. The residue obtained was washedwith n-pentane, filtered and dried to give the title compound (7 g,82%). MS (ESI) m/z 368 (M+H)⁺).

A stirred solution of toluene (50 mL),4-bromo-3-fluoro-N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)benzenesulfonamide(9 g, 24.6 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (9.33 g,36.7 mmol) and potassium acetate (7.23 g, 73.7 mmol) was degassed usingargon for 10 minutes.[1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.8 g, 2.5mmol) was added and the reaction mixture was degassed for another 10minutes and stirred at 110° C. for 4 hours. The reaction mixture wascooled to room temperature and filtered through celite. The organiclayer was separated, dried over sodium sulfate and concentrated underreduced pressure to give the title compound (10 g, 98%). For large scalebatches, the compound was used without further purification. When thispreparation was performed on a smaller scale, the residue was taken intoether, filtered and the filtrate was concentrated to give the desiredproduct. MS (ESI) m/z 412 (M−H)⁺).

A stirred solution of dioxane (50 mL),3-fluoro-N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(18 g, 43.6 mmol), 2-bromo-3,5-difluoropyridine (12.68 g, 65.37 mmol)and caesium carbonate (35.52 g, 109.0 mmol) was degassed using argon for10 minutes. [1,1-Bis(diphenylphosphino) ferrocene]dichloropalladium(II)(3.2 g, 4.4 mmol) was added and the reaction mixture was degassed foranother 10 minutes and then stirred at 110° C. for 6 hours. The solventwas evaporated under reduced pressure and the compound was extractedinto ethyl acetate. The organic layer was separated, dried over sodiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography followed by washing withn-pentane to give the title compound (8.19 g, 47%).

¹H NMR (400 MHz, methanol-d₄) δ 8.55 (d, J=2.1 Hz, 1H), 7.87-7.71 (m,4H), 3.24 (m, 1H), 1.8-1.7 (m, 2H), 1.66-1.55 (m, 2H), 1.51-1.34 (m,4H), 1.19 (s, 3H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2.5 mins, held at 95:5 B:A for 0.5 min, reduced linearly to30:70 B:A over 1 min and held at 30:70 B:A for the final 0.5 min.Retention time 1.88 min, m/z 399 (M−H)⁺.

A solution of3-fluoro-N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(0.338 g, 0.82 mmol) in dimethoxyethane (10 mL) was purged with argonfor 15 min. 2-bromo-3,5-dichloropyridine (0.185 g, 0.82 mmol) and sodiumcarbonate (0.175 g, 1.65 mmol) in water were added and degassed usingargon for 30 minutes. [1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.12 g, 0.16 mmol) was added and the reactionmixture was degassed for another 10 minutes and then stirred at 80° C.for 2 hours. The reaction mixture was cooled and filtered throughcelite. The solvent was evaporated under reduced pressure and theresidue was purified by silica gel column chromatography using 50% ethylacetate in hexane to give the title compound (0.04 g, 11%).

¹H NMR (400 MHz, methanol-d₄) δ 1.19 (3H, s), 1.33-1.52 (4H, m),1.54-1.66 (2H, m), 1.69-1.82 (2H, m), 3.25 (1H, m), 7.68 (1H, m), 7.75(1H, m), 7.82 (1H, m), 8.20 (1H, d, J=2.1 Hz), 8.65 (1H, d, J=2.1 Hz).

LCMS: mobile phase A: 10 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.2 mL/min. Run time: 5mins—starting solvent 35:65 B:A is increased linearly to 95:5 B:A overthe first 2.5 mins, held at 95:5 B:A for 1.3 min, reduced immediately to35:65 B:A for the final 1.2 min. Retention time 2.59 min m/z 431 (M−H)⁺.

A solution ofN-((1s,4s)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(17 g, 43.0 mmol), 2-bromo-5-chlorobenzonitrile (14 g, 64.7 mmol), andsodium carbonate (9.1 g, 86 mmol) in dioxane: water (240 mL, 9:1) wasdegassed using argon for 10 min.[1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (3.14 g, 4.3mmol) was added and the reaction mixture was degassed for another 10min. The reaction mixture was heated at 110° C. for 6 hours. Solvent wasevaporated under reduced pressure, water was added, and the compound wasextracted in ethyl acetate. The organic layer was separated, dried oversodium sulphate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography using 100-200 mesh silicagel using 20-40% ethyl acetate in hexane as eluent. The fractions wereconcentrated to 1/10^(th) the volume (85 mL) and then filtered. Theresidue obtained was washed with 20% ethyl acetate in hexane followed byn-pentane to afford the title compound. Yield: 5.8 g, 33% (over 2steps).

¹H NMR (400 MHz, Chloroform-d) δ 8.04-7.97 (m, 2H), 7.79 (d, J=2.2 Hz,1H), 7.69-7.64 (m, 3H), 7.48 (d, J=8.4 Hz, 1H), 4.45 (d, J=8 Hz, 1H),3.3-3.15 (br, 1H), 1.74-1.51 (m, 6H), 1.45-1.35 (m, 2H), 1.20 (s, 3H),1.00 (s, 1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.62 min m/z 403.30 [M−1].

A solution ofN-((1s,4s)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(13.5 g, 34.1 mmol), 1-bromo-4-fluoro-2-(trifluoromethyl)benzene (12.45g, 51.2 mmol), and sodium carbonate (7.24 g, 68.3 mmol) in dioxane:water (230 mL, 9:1) was degassed using argon for 10 min.[1,1-Bis(diphenylphosphino)ferrocene] dichloropalladium(II) (2.49 g, 3.4mmol) was added and the reaction mixture was degassed for another 10min. The reaction mixture was heated at 110° C. for 6 hours. Solvent wasevaporated under reduced pressure, water was added, and the compound wasextracted in ethyl acetate. The organic layer was separated, dried oversodium sulphate, and concentrated under reduced pressure. The residuewas purified by silica gel column chromatography using 100-200 meshsilica gel using 20-40% ethyl acetate in hexane as eluent. The fractionswere concentrated to 1/10^(th) of the volume (80 mL) and then filtered.The residue obtained was washed with 20% ethyl acetate in hexanefollowed by n-pentane to afford the title compound. Yield: 5.6 g, 20%(over 2 steps).

¹H NMR (400 MHz, Chloroform-d) δ 7.97-7.88 (m, 2H), 7.52-7.40 (m, 3H),7.31 (dd, J=7.0, 2.4 Hz, 2H), 4.40 (d, J=8.0 Hz, 1H), 3.25-3.12 (br,1H), 1.70-1.50 (m, 6H), 1.45-1.33 (m, 2H), 1.20 (s, 3H), 1.00 (bs, 1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.79 min m/z 430 [M−1].

A solution ofN-((1s,4s)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(15 g, 37.9 mmol), 2-bromo-1,3,5-trifluorobenzene (12 g, 56.9 mmol), andsodium carbonate (8.03 g, 75.8 mmol) in dioxane: water (250 mL, 9:1) wasdegassed using argon for 10 min.[1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (2.77 g, 3.8mmol) was added and the reaction mixture was degassed for another 10min. The reaction mixture was heated at 110° C. for 6 hours. Solvent wasevaporated under reduced pressure, water was added, and the compound wasextracted in ethyl acetate. The organic layer was separated, dried oversodium sulphate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography using 100-200 mesh silicagel using 20-40% ethyl acetate in hexane as eluent. The fractions wereconcentrated to 1/10^(th) the volume (90 mL) and then filtered. Theresidue obtained was washed with 20% ethyl acetate in hexane followed byn-pentane to afford the title compound. Yield: 4.98 g, 18% (over 2steps).

¹H NMR (400 MHz, Chloroform-d) δ 7.99-7.92 (m, 2H), 7.61-7.53 (m, 2H),6.80 (t, J=8.2 Hz, 2H), 4.39 (d, J=7.8 Hz, 1H), 3.25-3.12 (br, 1H),1.75-1.51 (m, 6H), 1.48-1.33 (m, 2H), 1.20 (s, 3H), 0.99 (s, 1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.65 min m/z 398 [M−1].

A solution of3-fluoro-N-((1s,4s)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(3.1 g, 7.5 mmol), 1-bromo-2,4-difluorobenzene (2.1 g, 10.9 mmol), andsodium carbonate (1.6 g, 15.0 mmol) in dioxane:water (60 mL, 5:1) wasdegassed using argon for 10 min. [1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.548 g, 0.75 mmol) was added and the reactionmixture was degassed for another 10 min. The reaction mixture was heatedat 110° C. for 6 hours. Solvent was evaporated under reduced pressure,water was added, and the compound was extracted in ethyl acetate. Theorganic layer was separated, dried over sodium sulphate and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography using 100-200 mesh silica gel with 20-40% ethyl acetatein hexane as eluent to afford the title compound. Yield: 0.6 g, 18%(over 2 steps).

¹H NMR (400 MHz, Chloroform-d) δ 7.73-7.69 (m, 2H), 7.56-7.47 (m, 1H),7.44-7.32 (m, 1H), 7.06-6.91 (m, 2H), 4.41 (d, J=7.8 Hz, 1H), 3.27-3.14(br, 1H), 1.76-1.52 (m, 6H), 1.48-1.35 (m, 2H), 1.21 (s, 3H), 0.97 (s,1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.70 min m/z 398 [M−1].

A solution of3-fluoro-N-((1s,4s)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(22 g, 53.2 mmol), 2-bromo-3,5-difluoropyridine (15.5 g, 79.9 mmol), andcesium carbonate (52.0 g, 159.6 mmol) in dioxane: water (100 mL) wasdegassed using argon for 10 min. [1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (3.9 g, 5.3 mmol) was added and the reactionmixture was degassed for another 10 min. The reaction mixture was heatedat 110° C. for 6 hours. Solvent was evaporated under reduced pressure,water was added, and the compound was extracted in ethyl acetate. Theorganic layer was separated, dried over sodium sulphate and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography using 100-200 mesh silica gel using 20-40% ethyl acetatein hexane as eluent. The fractions were concentrated to 1/10^(th) thevolume (110 mL) and filtered. The residue obtained was washed with 20%ethyl acetate in hexane followed by n-pentane to afford the titlecompound. Yield: 5.6 g, 26% (over 2 steps).

¹H NMR (400 MHz, Chloroform-d) δ 8.50 (m, 1H), 7.83-7.67 (m, 3H),7.41-7.33 (m, 1H), 4.43 (d, J=7.8 Hz, 1H), 3.28-3.13 (m, 1H), 1.74-1.52(m, 6H), 1.46-1.35 (m, 2H), 1.21 (s, 3H), 0.97 (s, 1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.42 min m/z 399 [M−1].

To a stirred solution ofN-((1s,4s)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(26.5 g, 67.0 mmol), 2-bromo-3,5-difluoropyridine (19.5 g, 100.5 mmol),and sodium carbonate (14.22 g, 134.2 mmol) in dioxane: water (250 mL,9:1) was degassed using argon for 10 min.[1,1-Bis(diphenylphosphino)ferrocene] dichloropalladium(II) (4.90 g,6.70 mmol) was added and the reaction mixture was degassed for another10 min. The reaction mixture was heated at 110° C. for 6 hours. Solventwas evaporated under reduced pressure, water was added, and the compoundwas extracted in ethyl acetate. The organic layer was separated, driedover sodium sulphate and concentrated under reduced pressure to obtain aresidue. The residue was purified by silica gel column chromatographyusing 100-200 mesh silica gel using 20-40% ethyl acetate in hexane aseluent. The fractions were concentrated to 1/10^(th) of the volume (100mL) and then filtered. The residue obtained was washed with 20% ethylacetate in hexane followed by n-pentane to afford the title compound.Yield: 5.9 g, 26% (over 2 steps).

¹H NMR (400 MHz, Chloroform-d) δ 8.48 (d, J=2.4 Hz, 1H), 8.13-8.06 (m,2H), 7.98 (d, J=8.5 Hz, 2H), 7.39-7.31 (m, 1H), 4.39 (d, J=7.8 Hz, 1H),3.25-3.1 (br, 1H), 1.72-1.5 (m, 6H), 1.42-1.32 (m, 2H), 1.19 (s, 3H),0.96 (s, 1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.41 min m/z 381 [M−1].

A solution ofN-((1s,4s)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(1.5 g, 3.8 mmol), 2-bromo-3,5-dichloropyridine (1.3 g, 5.7 mmol), andsodium carbonate (0.805 g, 7.6 mmol) in dioxane: water (18 mL, 5:1) wasdegassed using argon for 10 min.[1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.277 mg,0.38 mmol) was added and the reaction mixture was degassed for another10 min. The reaction mixture was heated at 110° C. for 6 hours. Afurther solution ofN-((1s,4s)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(4 g, 10.1 mmol), 2-bromo-3,5-dichloropyridine (3.44 g, 15.2 mmol), andsodium carbonate (2.14 g, 20.2 mmol) in dioxane:water (54 mL, 5:1) wasdegassed using argon for 10 min. [1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.739 g, 1.0 mmol) was added and the reactionmixture was degassed for another 10 min. The reaction mixture was heatedat 110° C. for 6 hours. Both the above batches were combined. Solventwas evaporated under reduced pressure, water was added, and the compoundwas extracted in ethyl acetate. The organic layer was separated, driedover sodium sulphate and concentrated under reduced pressure to obtain aresidue. The residue was purified by silica gel column chromatographyusing 100-200 mesh silica gel with 20-40% ethyl acetate in hexane as theeluent. The fractions were concentrated to 1/10^(th) of the volume (30mL) and then filtered. The residue obtained was washed with 20% ethylacetate in hexane followed by n-pentane to afford the title compound.Yield: 0.79 g, 14% (over 2 steps).

¹H NMR (400 MHz, Chloroform-d) δ 8.59 (m, 1H), 8.02-7.94 (m, 2H),7.90-7.83 (m, 3H), 4.47 (d, J=8 Hz, 1H), 3.25-3.1 (br, 1H), 1.73-1.5 (m,6H), 1.44-1.32 (m, 2H), 1.19 (s, 3H), 1.01 (s, 1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.69 min m/z 413[M−1].

A solution of3-fluoro-N-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(3.5 g, 8.4 mmol), 1-bromo-2,4-difluorobenzene (2.44 g, 12.7 mmol), andsodium carbonate (1.79 g, 16.9 mmol) in dioxane: water (60 mL, 5:1) wasdegassed using argon for 10 min. [1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.619 g, 0.85 mmol) was added and the reactionmixture was degassed for another 10 min. The reaction mixture was heatedat 110° C. for 6 hours. Solvent was evaporated under reduced pressure,water was added, and the compound was extracted in ethyl acetate. Theorganic layer was separated, dried over sodium sulphate, andconcentrated under reduced pressure. The residue was purified by SFCpurification: Mobile Phase: CO2:Methanol (05-50 in 5 min), Column:Silica 2-Ethylpyridine (250×4.6 mm, 5μ), Flow: 3 mL/min, Wavelength:210-400 nm to afford the title compound. Yield: 0.7 g, 19% (over 2steps).

¹H NMR (400 MHz, Chloroform-d) δ 7.78-7.64 (m, 2H), 7.62-7.45 (m, 1H),7.44-7.34 (m, 1H), 7.06-6.91 (m, 2H), 4.51 (d, J=6.8 Hz, 1H), 3.40-3.35(br, 1H), 1.95-1.83 (m, 2H), 1.67-1.37 (m, 6H), 1.25 (d, J=3.3 Hz, 3H),1.13 (s, 1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 4.5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.56 min m/z 398 [M−1].

2-Bromo-5-chloro-benzonitrile (297.0 g, 1.372 mol), phenyl boronic acid(184.0 g, 1.509 mol), sodium carbonate (436.3 g, 4.116 mol),1,2-dimethoxyethane (4455 mL) and water (1485 mL) were added to a vesselunder nitrogen. The flask was degassed three times with nitrogen andtetrakis(triphenylphosphine)palladium(0) (79.3 g, 0.069 mol) was added.The flask was degassed three times with nitrogen and was stirred andheated to 70° C. and then stirred at that temperature for 24 h. Furtherphenyl boronic acid (36.8 g, 0.302 mol), sodium carbonate (87.3 g, 0.824mol) and tetrakis(triphenylphosphine) palladium (15.9 g, 0.014 mol) wereadded and the mixture stirred for a further 16 h. The reaction wascooled to room temperature and the suspension was filtered and the solidwashed with ethyl acetate (2×2000 mL). The combined filtrates wereseparated and the organic layer was washed with saturated brine (2×2000mL). The organic layer was dried over anhydrous magnesium sulphate,filtered and the solid washed with ethyl acetate (1000 mL). The combinedfiltrates were concentrated whilst absorbing onto silica gel (600 g).The crude material was purified on silica gel (6 Kg) eluting with 25-50%toluene in heptane. The pure fractions were combined and concentrated togive a white solid. The solid was azeotroped with heptane (8×800 mL) toremove any residual toluene and give the target compound. Yield=215.9 g(73.6%).

1H NMR (270 MHz, DMSO d₆) δ 8.18-8.12 (m, 1H), 7.86 (dd, J=8.4 Hz, 2.4Hz, 1H), 7.7-7.45 (m, 6H).

To a 5 L flask was charged crude 5-chloro-2-phenylbenzonitrile (407.8 g,1.909 mol) and chloroform (2325 mL). The pale yellow solution was cooledto less than 5° C. and chlorosulfonic acid (343 mL, 5.15 mol) was added,keeping the temperature at less than 10° C. The reaction mixture wasallowed to warm to room temperature and the dark brown solution wasstirred at room temperature overnight. The reaction mixture wasconcentrated at 20° C. and the residue was dissolved in ethyl acetate(2325 mL)-exotherm to 33° C. observed. Water (490 mL) was added(exotherm to 64° C.) followed by saturated brine (1920 mL), and themixture was cooled to 19° C. The thick suspension was filtered (veryslow filtration) and the residue washed with water (2×1 L) and ethylacetate (2×1.5 L). The filter cake was dried in vacuo at 40° C. for 64hours to afford 435 g of material as a yellow solid. This was slurriedin ethyl acetate (1740 mL) at room temperature for 20 minutes. Thesuspension was filtered and the residue washed with ethyl acetate (435mL). The filter cake was dried in vacuo at 40-60° C. over two nights toafford a cream solid (366.3 g), containing 289 g of the title compound(52% yield). Analysis by Karl Fischer showed the product contained 4.3%water.

¹H NMR (270 MHz, DMSO d₆) δ 8.16 (d, J=2.3 Hz, 1H), 7.85 (dd, J=2.3 Hz,8.5 Hz, 1H), 7.73 (d, J=8.2 Hz, 2H), 7.65 (d, J=8.2 Hz. 1H), 7.54 (d,J=8.3 Hz, 2H).

To a 5 L flask was charged crude4-(4-chloro-2-cyanophenyl)benzene-1-sulfonic acid (366.3 g, containing289 g 4-(4-chloro-2-cyanophenyl)benzene-1-sulfonic acid, 0.98 mol) andtoluene (3000 mL). Thionyl chloride (423 mL, 5.83 mol) was addeddropwise followed by dimethylformamide (5 mL, 0.0646 mol). The reactionmixture was heated to 75° C. and stirred overnight. The reaction mixturewas cooled, concentrated in vacuo, and the residue was partitionedbetween ethyl acetate (3000 mL) and water (1500 mL) and the organiclayer washed with saturated brine (1500 mL). The organic layer was driedover anhydrous magnesium sulphate (50 g) and filtered. The residue waswashed with ethyl acetate (2×100 mL) and the combined organic layersconcentrated to afford the title compound (297.6 g, 93% yield) as ayellow solid. Analysis by NMR indicated a purity of 95%.

¹H NMR (270 MHz, DMSO d₆) δ 8.16 (d, J=2 Hz, 1H), 7.87 (dd, J=2.1 Hz,8.2 Hz, 1H), 7.73 (d, J=8.2 Hz, 2H), 7.64 (d, J=8.2 Hz. 1H), 7.54 (d,J=8.3 Hz, 2H).

A stirred solution ofN-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(1.6 g, 4.05 mmol), 2-bromo-5-fluorobenzonitrile (2.03 g, 10.15 mmol),sodium carbonate (1.07 g, 10.1 mmol) in dioxane: water (30:3 mL) wasdegassed using argon for 10 min.[1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.296 g,0.405 mmol) was added and the reaction mixture was degassed for another10 min and stirred at 110° C. for 6 h. Solvent was evaporated underreduced pressure and the compound was extracted in ethyl acetate. Theorganic layer was separated, dried over sodium sulphate and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography. The obtained residue was washed with hexane followed byn-pentane to afford the title compound as a white solid. Yield: 1.1 g,70%.

¹H NMR (400 MHz, Chloroform-d) δ 8.04-7.97 (m, 2H), 7.71-7.64 (m, 2H),7.55-7.49 (m, 2H), 7.39-7.46 (m, 1H), 4.46 (d, J=6.6 Hz, 1H), 3.45-3.32(m, 1H), 1.92-1.82 (m, 2H), 1.65-1.35 (m, 6H), 1.23 (s, 3H), 1.11 (brs,1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2.50 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.50 min (ESI) m/z 387[M−1].

A stirred solution ofN-((1r,4r)-4-hydroxy-4-methylcyclohexyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide(1.6 g, 4.05 mmol), 4-bromo-3-fluorobenzonitrile (2.03 g, 10.1 mmol),sodium carbonate (1.07 g, 10.1 mmol) in dioxane/water (30/3 mL) wasdegassed using argon for 10 min.[1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.296 g,0.405 mmol) was added and the reaction mixture was degassed for another10 min and stirred at 110° C. for 6 h. Solvent was evaporated underreduced pressure and the compound was extracted in ethyl acetate. Theorganic layer was separated, dried over sodium sulphate and concentratedunder reduced pressure. The residue was purified by flash silica gelcolumn chromatography. The residue obtained was washed with hexanefollowed by n-pentane to give the title compound as a white solid.Yield: 1.19 g, 75.79%.

¹H NMR (400 MHz, Chloroform-d) δ 8.03-7.96 (m, 2H), 7.73-7.66 (m, 2H),7.62-7.56 (m, 2H), 7.54-7.48 (m, 1H), 4.51 (d, J=6.65 Hz, 1H), 3.42-3.30(m, 1H), 1.95-1.82 (m, 2H), 1.66-1.55 (m, 2H) 1.53-1.37 (m, 4H), 1.23(s, 3H), 1.12 (brs, 1H).

LCMS: mobile phase A: 5 mM ammonium formate in water+0.1% ammonia,mobile phase B: acetonitrile+5% mobile phase A+0.1% ammonia; Column: YMCTriart, C18 (50×4.6 mm) 3 uM; Flow rate: 1.4 mL/min. Run time: 5mins—starting solvent 30:70 B:A is increased linearly to 95:5 B:A overthe first 2.50 mins, held at 95:5 B:A for 1.5 min, reduced linearly to30:70 B:A over 0.5 min and held at 30:70 B:A for the final 0.5 min.Retention time 2.52 min (ESI) m/z 387[M−1].

Additional Compounds

The following compounds were also prepared for use as referencecompounds in the biological studies described herein:

Code Structure ABD599

ABD735

ABD836

ABD899

ABD900

REF001

Biological Studies

Potency was assessed using a viability assay based on the survival ofthe J774 macrophage cell line. Macrophages are closely related toosteoclasts and have been used previously as a model system forosteoclast survival (see, e.g., Luckman et al., 1998,“Heterocycle-containing bisphosphonates cause apoptosis and inhibit boneresorption by preventing protein prenylation: evidence fromstructure-activity relationships in J774 macrophages,” J. Bone Miner.Res., Vol. 13, pp. 1668-1678). The model is indicative both of effectson bone protection in diseases such as osteoporosis, osteoarthritis andrheumatoid arthritis, and of effects on inflammation since, likeosteoclasts, J774 macrophages are dependent for survival on continuedNFκB activation.

Metabolic stability was measured by determining the rate ofdisappearance of compound in the presence of human liver microsomalpreparations, as quantified by liquid chromatography mass-spectrometryand standard mass-spectrometry (LC-MS/MS).

Solubility was measured by equilibration of the compound in fasted statesimulated intestinal fluid (FaSSIF) and quantified by high-performanceliquid chromatography (HPLC).

Anti-inflammatory effects were further characterised by assessing theproduction of interleukin-6 (IL-6) by human Thp-1 derived macrophagesstimulated with a pro-inflammatory stimulus bacterial lipopolysaccharide(LPS). LPS acts with a cell-surface receptor, Toll-like receptor-4 toactivate the NFκB and IRF signalling pathways to produce IL-6. Thereduction of IL- in this stimulated assay is indicative ofanti-inflammatory effects with utility in the treatment of conditions inwhich IL-6 production is aberrant.

In vivo studies were also carried out to evaluate the potential of thesecompounds as drugs.

Pharmacokinetics were assessed in rats and effects on disease wereassessed in a mouse model of collagen-induced arthritis.

Biological Study 1 Resazurin Macrophage J774 Viability Assay

In vitro potency of test compounds was determined by incubation withJ774 macrophages and subsequent determination of cell viability usingresazurin.

Resazurin is a redox dye commonly used as an indicator of viability incultured cells (see, e.g., Anoopkumar-Dukie, 2005, British Journal ofRadiology, Vol. 78, pp. 945-947). It is non-toxic to cells and stable inculture medium, allowing continuous measurement of cell proliferation invitro as either a kinetic or endpoint assay. The assay is based on theability of viable, metabolically-active cells to reduce resazurin (whichis blue and non-fluorescent) to resorufin and dihydroresorufin (whichare red and fluorescent) using electrons from reducing species, such asnicotinamide adenine dinucleotide (NADPH) and flavin adeninedinucleotide (FADH). This transformation, from oxidised form to reducedform, can be measured either colorimetrically or fluorometrically.Insults that impair cell viability and proliferation also affects thecapacity of cells to reduce resazurin, and the rate of dye reduction isdirectly proportional to the number of viable cells present.

For fluorescence measurements, 530-560 nm excitation and 590 nm emissionwavelengths are typically used. For colorimetric measurements,absorbance at 570 nm (reduced form) and 600 nm (oxidised form) istypically measured. A simple calculation is performed to determine therelative quantities of the two species: a high ratio of resorufin (thereduced form) to resazurin (the oxidised form) is an indicator thatcells are proliferating and viable. A low ratio indicates cells that arequiescent or non-viable.

J774 cells were plated at 10⁴ cells per well in 100 μL αMEM (α ModifiedEagle Medium) in 96-well plates and allowed to adhere overnight. Thefollowing day, test compounds were prepared as 100 mM solutions in DMSO.These stock solutions were diluted in DMSO and then diluted 1000× inculture medium (aMEM) before being added directly to the wells so as togive the desired final compound concentration. After a 72 hourincubation at 37° C./5% CO₂, resazurin (Alamar Blue, BiosourceInternational) was added to each well (1:10 v/v, 10 μL). The plate wasthen incubated at 37° C. for 3 hours and fluorescence was measured at590 nm, with a 25 nm bandwidth.

The average results for each test compound were expressed as a percent(%) of the average control value reflecting cell viability. The averagevalues across the concentrations tested were then plotted and the IC₅₀for was calculated by fitting the data to a 4-parameter IC₅₀ equationusing software from Grafit (Erithacus Software). Each experiment wasrepeated twice and the data are presented as the mean IC₅₀ from bothexperiments.

The results are summarised in the following table.

TABLE 1 Resazurin Macrophage J774 Viability Assay Compound IC₅₀ (μM) ⁽¹⁾IC₅₀ (μM) ⁽²⁾ ABD599 0.19 0.41 ABD735 0.10 0.07 ABD836 0.28 1.45 ABD9000.56 3.64 ABD899 0.08 0.27 REF001 0.12 0.05 HMC-C-01-A 0.13 1.89HMC-C-02-A 0.26 14.7 HMC-C-03-A 0.14 0.41 HMC-C-04-A 0.50 HMC-C-05-A2.46 HMC-C-06-A 0.71 HMC-N-01-A 0.13 2.66 HMC-N-02-A 0.18 4.59HMC-N-03-A 0.17 0.62 HMC-N-04-A 0.18 HMC-C-01-B 0.16 HMC-C-02-B 1.74HMC-C-03-B 0.20 HMC-C-04-B 0.06 HMC-N-01-B 0.14 HMC-N-02-B 0.36HMC-N-03-B 0.08 ⁽¹⁾ Results from a resazurin macrophage viability assayconducted in a 6 point concentration range from 10 μM to 10 nM with n =3 replicates per concentration. Data are the mean from 2 independentexperiments. ⁽²⁾ Results from a resazurin macrophage viability assayconducted in a 12 point concentration range from 10 μM to 0.5 nM with n= 4 replicates per concentration. Data are the mean from 3 independentexperiments.

These data demonstrate that the HMC compounds described herein, andparticularly HMC-C-01-A and HMC-C-01-B; HMC-C-03-A and HMC-C-03-B;HMC-C-04-A and HMC-C-04-B; HMC-C-06-A; HMC-N-01-B; HMC-N-02-B; andHMC-N-03-B show excellent potency in the resazurin macrophage viabilityassay and no loss of potency, as compared to the reference compounds.

Biological Study 2 Human Liver Microsomal Stability

Metabolic stability of test compounds was measured by determination ofthe rate of disappearance of the compound when incubated in the presenceof human liver microsomes. Liver microsomes are prepared from theendoplasmic reticulum of hepatocytes and are the primary source of themost important enzymes (cytochrome P450s) involved in drug metabolism.Study of drug stability in the presence of liver microsomes is acceptedas a valuable model permitting rapid prediction of in vivo drugstability.

Human liver microsomes were obtained from a commercial source. Testcompounds (1 μM) were incubated with pooled liver microsomes (male andfemale). Samples were incubated for a 60 minute period and removed at upto 6 time points and analysed by LC-MS/MS for the presence/amount oftest compounds.

Microsomes (final protein concentration 0.25 or 0.5 mg/mL), 0.1 Mphosphate buffer pH 7.4, and test compound (final concentration 1 μM;diluted from 10 mM stock solution to give a final DMSO concentration of0.1%) were incubated at 37° C. prior to the addition of Nicotinamideadenine dinucleotide phosphate (NADPH, final concentration 1 mM) toinitiate the reaction. The final incubation volume was 100 μL. A controlincubation was included for each compound tested, where 0.1 M phosphatebuffer pH 7.4 was added instead of NADPH. The positive control compoundterfenadine was included in each experiment and all incubations wereperformed once for each compound.

Each compound was incubated for 0, 5, 15, 30, 45 or 60 minutes. Thereactions were stopped by the addition of 100 μL ice-cold acetonitrilecontaining internal standard (0.001 mM glipizide) at the appropriatetime points. The incubation plates were centrifuged at 4000 rpm for 15minutes at 4° C. to precipitate the protein and 0.1 mL aliquots wereanalysed using LC-MS/MS, with the conditions shown in the followingtable.

TABLE 2 LC-MS/MS Conditions HPLC: Schimadzu Agilent MS/MS: API 4000, API4000 Q-Trap Software: Analyst 1.5 Ionisation mode: Turbo spray, positivemode ionisation Scan mode: Multiple reaction monitoring (MRM) Column:Waters, Xterra, MS-C18 (2) 5 μm 50 × 3.0 mm Column Temperature (° C.):40 Phase A: 0.1% formic acid in water Phase B: 0.1% formic acid inacetonitrile Standard Injections (μL): 1, 2, 3, 5, 7, 10 Test Injections(μL): 1, 2, 3, 10, 20, 50 Flow Rate (mL/min): 0.8-1

From a plot of the natural logarithm of the peak area ratio (i.e., theratio of compound peak area: internal standard peak area) against time,the gradient of the line was determined. Subsequently, half-life(t_(1/2)) and intrinsic clearance (CL_(int)) were calculated using thefollowing equations, where V=Incubation Volume (μL/mg microsomalprotein):Eliminated Rate Constant(k)=(−Gradient)Half-Life(t _(1/2))(min)=0.063/kIntrinsic Clearance(CL _(int))(μL/min/million cells)=(V×0.693)/t _(1/2)

The data are summarised in the following table.

TABLE 3 Human Liver Microsomal Stability Compound T_(1/2) (min) ⁽¹⁾T_(1/2) (min) ⁽²⁾ ABD599 287 ABD735 524 ABD836 >900 ABD899 72 105 ABD90087 REF001 43 HMC-C-01-A 82 HMC-C-02-A 55 HMC-C-03-A 84 HMC-C-04-A 100HMC-C-05-A 123 HMC-C-06-A 240 HMC-N-01-A 175 HMC-N-02-A 367 HMC-N-03-A258 HMC-N-04-A 156 HMC-C-01-B 70 HMC-C-02-B 18 HMC-C-03-B 48 HMC-C-04-B107 HMC-N-01-B 127 HMC-N-02-B 168 HMC-N-03-B 70 ⁽¹⁾ Compounds wereincubated with human liver microsomes at a final protein concentrationof 0.25 mg/mL with sampling at 5 time points: 0, 5, 15, 30, and 60minutes. One replicate was performed per time point. ⁽²⁾ Compounds wereincubated with human liver microsomes at a final protein concentrationof 0.5 mg/mL with sampling at 6 time points: 0, 5, 15, 30, 45 and 60minutes. Two replicates were performed per time point.

The data demonstrate that the HMC compounds described herein showmetabolic stability equivalent to that of the reference compounds.

Biological Study 3 Aqueous Solubility

Aqueous solubility was measured by equilibration of compounds withfasted state simulated intestinal fluid (FaSSIF) and quantifiedspectrophotometrically.

FaSSIF was prepared as described below:

Preparation of blank FaSSIF: 0.21 g of sodium hydroxide (NaOH) pellets,1.97 g of dihydrogen sodium phosphate (NaH₂PO₄.2H₂O) and 3.09 g ofsodium chloride (NaCl) were dissolved in 400 mL of deionised water. ThepH was adjusted to 6.5 using 1 M hydrochloric acid and further deionisedwater added to a final volume of 500 mL.

Preparation of FaSSIF: 0.056 g of SIF Powder (containing sodiumtaurocholate and lecithin) (Phares AG) was dissolved in 25 mL of blankFaSSIF and stirred until the powder was completely dissolved. Thesolution was allowed to stand for 2 hours during which it becameopalescent; it was used within 24 hours. The final solution compositionwas characterised as follows:

-   -   Sodium taurocholate: 3 mM    -   Lecithin: 0.75 mM    -   Osmolarity: 270±10 mOsmol    -   pH: 6.5

Aqueous solubility was determined by spiking a known concentration oftest compound (dissolved in DMSO) into FaSSIF followed by incubation for16 hours. The optical density was measured at the end of the incubationperiod for test compounds and a reference used to determine solubility.In brief, two samples were prepared for each determination: a referencesample consisting of a stock solution of test compound in DMSO dilutedin system solution (a phosphate free, low absorption buffer) andpropanol; and a test sample (prepared in triplicate) consisting of 0.5mL FaSSIF spiked with test compound at 0.2 mM. Each sample was incubatedat room temperature for 16 hours with constant shaking at 250 rpm. Atthe end of the incubation period, 0.3 mL of each sample was filteredthrough a pION filter plate (PION, Woburn Mass.), diluted 1:1 withpropanol and scanned using UV spectrophotometry at λ_(max) (190-400 nM)using a Spectra Max Plus-Version 2.1000 (Molecular Devices, Sunnyvale,Calif.), with μSOL Explorer solubility determination software (pION,Woburn, Mass.).

FaSSIF solubility was calculated using the following formula:

${{FaSSIF}\mspace{14mu}{Solubility}},{\frac{mg}{mL} = \frac{\left\lbrack \frac{150}{75} \right\rbrack*\left\lbrack \frac{{OD}\mspace{14mu}{of}\mspace{14mu}{sample}}{{OD}\mspace{14mu}{of}\mspace{14mu}{reference}} \right\rbrack*{Cr}*{molecular}\mspace{14mu}{weight}}{10^{6}}}$wherein:“OD” is the optical density;“Cr” is the concentration of the reference (33.4 μM); and“molecular weight” is for the test compound (e.g., 381.44 for ABD735).

The data are summarised in the following table.

TABLE 4 FaSSIF Solubility Compound Solubility (mg/mL) ⁽¹⁾ Solubility(mg/mL) ⁽²⁾ ABD599 0.03 ABD735 0.02 ABD836 0.03 ABD899 0.06 0.13 ABD9000.12 REF001 0.05 HMC-C-01-A 0.06 HMC-C-02-A 0.04 HMC-C-03-A 0.03HMC-C-04-A 0.08 HMC-C-05-A 0.19 HMC-C-06-A 0.11 HMC-N-01-A 0.03HMC-N-02-A 0.02 HMC-N-03-A >0.08 HMC-N-04-A 0.06 HMC-C-01-B 0.08HMC-C-02-B 0.07 HMC-C-03-B 0.15 HMC-C-04-B 0.13 HMC-N-01-B 0.12HMC-N-02-B 0.12 HMC-N-03-B 0.10 ⁽¹⁾ Three replicates were run per studyat pH 6.5. ⁽²⁾ Two replicates were run per study at pH 6.8.

The data demonstrate that the HMC compounds described herein showsolubility equivalent to that of the reference compounds.

Biological Study 4 Thp1 Macrophage IL-6 Release Assay

In vitro potency of test compounds in human cells was determined byincubation with Thp1 macrophages and subsequent stimulation with aninflammatory stimulus (bacterial lipopolysaccharide (LPS)) followed bymeasurement of cellular interleukin-6 (IL-6) release.

The assay is strongly indicative of effects on inflammation. LPS is aligand for Toll-like receptor-4 (TLR4), which is a member of theToll-like receptor family of cell surface receptors. This receptor isimportant in the activation of the innate immune system, the majorfunctions of which are to:

-   -   (a) recruit immune cells to sites of infection through the        production of cytokines such as IL-6;    -   (b) activate the complement cascade, to identify bacteria,        activate cells and clear both dead cells and antibody complexes;    -   (c) activate the removal of foreign substances by cells such as        macrophages and dendritic cells; and    -   (d) activate antigen presentation, part of the adaptive immune        system.

TLR4 exerts its effects by activating a signalling cascade that resultsin the activation of several transcription factors including NFκB andmembers 3, 5, and 7 of the interferon regulatory transcription factor(IRF) family (IRF-3, IRF-5, and IRF-7). The activation of thesetranscription factors, and particularly NFκB and IRF-5 drives thesynthesis and secretion of cytokines such as interleukin 6 (IL-6).

Over-production/expression of IL-6 is associated with a range ofdisorders, including autoimmunity, inflammatory and cancer. IL-6 ispredominantly synthesised by macrophages and T-cells and is heavilyinvolved in governing the transition from acute to chronic inflammation.It does this by modifying the composition of the white blood cellinfiltrate in the inflammatory space, moving it from neutrophils tomonocyte/macrophages (see, e.g., Gabay, 2006). In addition, IL-6 exertsstimulatory effects on T- and B-cells (thus favouring chronicinflammatory responses) as well as on osteoclasts (thus promoting theturnover of bone). These effects are involved in the pathology ofseveral diseases including osteoporosis, rheumatoid arthritis, diabetes,atherosclerosis, depression, Alzheimer's disease, systemic lupuserythematosus, Behçet's disease, multiple myeloma, and prostate cancer.Furthermore, patients with advanced or metastatic cancer have higherthan normal circulating levels of IL-6. Decreasing IL-6 levels inmacrophages is therefore therapeutically beneficial.

Thp1 cells were plated at a concentration of 1×10⁵ cells/well or 1.7×10⁵cells/well in 500 μL or 150 μL RPMI complete media containing 1%penicillin-streptomycin and 10% heat inactivated foetal bovine serum in24-well plates or 96-well plates, respectively and allowed to adhereovernight. The following day, the cells were stimulated with phorbolmyristic acid (PMA) at a final concentration of 100 nM (24-well plates)or 200 nM (96-well plates) to induce differentiation and maintained forup to 8 days with a medium change at 5 days if cells were cultured to 8days. Test compounds were prepared as 100 nM solutions in DMSO and thenserially diluted in DMSO prior to dilution in culture medium. Thediluted compounds were added to the cultures 1 hour prior to stimulationwith 100 ng/mL LPS. Following a 16 or 18 hour incubation at 37° C./5%CO₂, the cell culture medium was collected and assayed for human IL-6levels using the human IL-6 duo-set ELISA kit (R&D Systems). The averageresults for each test compound (n=3) were expressed as a percent (%) ofthe average control value. The average values across the concentrationstested were then plotted and the IC₅₀ for the inhibition of IL-6 wascalculated by fitting the data to a 4-parameter IC₅₀ equation usingsoftware from Grafit version 6.0.12 (Erithacus Software Ltd., by DrRobin Leatherbarrow) or GraphPad Prism version 5.04 for Windows(GraphPad Software, La Jolla, Calif., USA, www.araphpad.com). Eachexperiment was repeated twice and the data are presented as the meanIC₅₀ from both experiments.

The results are summarised in the following table.

TABLE 5 Macrophage IL-6 Release Assay Data Compound IC₅₀ (μM) ⁽¹⁾ IC₅₀(μM) ⁽²⁾ ABD599 0.07 ABD899 0.03 ABD900 0.09 HMC-C-01-A 0.04 0.18HMC-C-02-A 0.001 0.97 HMC-C-03-A 0.004 0.05 HMC-C-04-A 0.13 HMC-C-05-A1.02 HMC-C-06-A 0.19 HMC-N-01-A 0.13 0.28 HMC-N-02-A 0.15 0.29HMC-N-03-A 0.05 HMC-C-01-B 0.06 HMC-C-02-B 0.29 HMC-C-03-B 0.02HMC-C-04-B 0.03 HMC-N-01-B 0.03 HMC-N-02-B 0.02 HMC-N-03-B 0.02 ⁽¹⁾ Thp1cells were plated at a concentration of 1 × 10⁵ cells/well in 500 μLRPMI complete media containing 1% penicillin-streptomycin and 10% heatinactivated foetal bovine serum in 24-well plates for 8 days with amedium change at 5 days. Compounds were tested in triplicate in a 6point concentration response curve at concentrations of 10, 1, 0.1,0.01, 0.001 and 0.0001 μM, with IL6 levels measured 16 hours after LPSstimulation. IC₅₀'s were calculated using Grafit version 6.0.12(Erithacus Software). ⁽²⁾ Thp1 cells were plated at a concentration of1.7 × 10⁵ cells/well in 150 μL RPMI complete media containing 1%penicillin-streptomycin and 10% heat inactivated foetal bovine serum in96-well plates for 3 days. Compounds were tested in triplicate in a 9point concentration response curve at concentrations of 30, 10, 3, 1,0.3, 0.1, 0.03, 0.01 and 0.001 μM with IL6 levels measured 18 hoursafter LPS stimulation. IC₅₀'s were calculated using GraphPad Prismversion 5.04 for Windows (GraphPad Software).

These data demonstrate that the HMC compounds described herein showexcellent potency in inhibiting IL-6 release from human macrophages,indicating their utility in the treatment of disorders in which IL-6 isup-regulated. Compounds HMC-C-01-A, HMC-C-03-A, HMC-C-06-A, HMC-C-01-B,HMC-C-03-B, HMC-C-04-B, HMC-N-01-B, HMC-N-02-B and HMC-N-03-B showparticularly good activity in reducing IL-6 release.

Biological Study 5 Rodent Pharmacokinetics Studies

Absorption and metabolic stability were studied using an in vivopharmacokinetics assay.

Three male Sprague-Dawley rats, aged 8-12 weeks, were dosed with testcompounds administered either orally or intravenously (dose level of 1mg/kg body weight intravenous or 5 mg/kg body weight orally). Testcompounds were formulated in 0.5% carboxymethylcellulose (CMC)/0.1%Tween-80 for administration via the oral route, or in 5% DMSO/10%solutol in saline for administration via the intravenous route. Forcompound HMC-C-01-A the oral administration was formulated in 2%dimethylacetamide/20% hydroxypropyl-β-cyclodextrin in water. Animalswere given free access to food throughout the study except for fastingovernight and until 2 hours post dose on the day of dosing.

Blood samples were taken from the retro-orbital plexus at the followingtime points and placed in microtubes containing 20% K₂EDTA solution:

Oral Dosing: predose; 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post dose.

Intravenous Dosing: predose; 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24 hourspost dose.

Blood samples were centrifuged to obtain plasma, which was transferredto a separate container and frozen at −20° C.

For analysis, samples were thawed at room temperature and prepared byprotein precipitation with acetonitrile spiked with internal standard(500 ng/mL glipizide) in the ratio 1:4 with plasma. The samples werethen vortexed for 5 minutes and centrifuged for 10 minutes at 20,600× gat 4° C. 100 μL of the supernatant was collected for analysis.

The standard samples were prepared similarly, after spiking blank ratplasma samples with 10 μL of analyte.

The concentration of test compound in rat plasma samples was determinedusing LC-MS/MS, with the conditions shown in the following table.

TABLE 6 LC-MS/MS Conditions HPLC: Schimadzu Agilent MS/MS: API 4000Software: Analyst 1.5 Ionisation mode: Turbo spray, negative mode Scanmode: Multiple reaction monitoring (MRM) Column Waters, Xterra, MS-C18(2) 5 μm 50 × 3.0 mm; Discovery Grace Smart RP183μ, 150 × 2.1, 3 μM;Waters Symmetry Shelf C18 75 × 4.6, 3.5 μM; Agilent Zorbax XDB, 150 ×4.6, 5 μM Column Temperature 40 (° C.): Phase A: Acetonitrile Phase B:0.1% formic acid Flow Rate (mL/min): 0.8-1.2

The pharmacokinetic parameters for the test compounds were calculated byPhoenix WinNonlin version 6.3 (Pharsight Corp, CA) using standardnon-compartmental methods. Peak plasma concentrations (C_(max)) and timeof the peak plasma concentration (T_(max)) were the observed values. Thearea under the plasma concentration-time curve (AUC) was determined byuse of the linear trapezoidal rule up to the last measurableconcentration (AUC_(last)) and thereafter by extrapolation of theterminal elimination phase to infinity (AUC_(inf)). The terminalelimination rate constant (k_(el)) was determined by regression analysisof the linear terminal portion of the log plasma concentration-timecurve. The elimination phase half-life (t_(1/2)) was calculated as0.693/k_(el). The tentative oral bioavailability (F) was calculated bydividing the AUC (0-24 hours) after oral administration by the adjustedAUC (0-8 hours) after intravenous administration (i.e., F=AUC(p.o.)×Dose (i.v.)/AUC (i.v.)×Dose (p.o.)) and reported as a percentage(%).

The pharmacokinetic data are summarised in the following table.

TABLE 7 Pharmacokinetic data Bioavail, F i.v. AUC p.o. AUC T_(1/2)Compound (%) (ng/mL/min) (ng/mL/min) (h) ABD735 83 1081   8965 ‡ 3.8ABD836 55 2142 5927 5.3 ABD899 50 2133  10740 ‡ 10.8 REF001 50  963  4766 ‡ 7.2 HMC-C-01-A 89  900 4002 6.2 HMC-C-02-A 39  546 1069 3.2HMC-C-03-A 55 1427 3910 2.4 HMC-N-01-A 64  740 1408 13.4 HMC-N-02-A 43  3053 † 3303 6.3 HMC-N-03-A 8   5102 †  962 3.1 HMC-N-04-A 66 1279 42032.9 HMC-C-01-B 67 1539 5121 5.7 HMC-C-03-B 116   816 * 9432 8.3HMN-C-04-B 59   1589 * 9360 5.5 HMC-N-01-B 60 2824 8454 9.1 HMC-N-02-B66 1931 6412 6.0 HMC-N-03-B 84   1380 * 11609  7.4 † Dosed at 2 mg/kgintravenously. * Dosed at 0.5 mg/kg intravenously. ‡ Dosed at 10 mg/kgorally.

These data demonstrate that the HMC compounds described herein haveexcellent oral pharmacokinetic properties equivalent to those of thereference compounds. This makes them suitable for use as oral drugs.

Biological Study 6 Mouse Collagen-Induced Arthritis

Seven- to eight-week-old male DBA/1j mice were used for all procedures.Animals were housed in groups of 10, and were maintained at 21° C.±2° C.on a 12-hour light/dark cycle with food and water ad libitum. CompleteFreund's adjuvant (CFA) was prepared by emulsifying bovine type IIcollagen at 4 mg/mL with a 4 mg/mL suspension of Mycobacteriumtuberculosis H37Ra in Incomplete Freund's adjuvant (IFA) (0.85 mLparaffin oil and 0.15 mL mannide monooleate) in a 1:1 (v/v) ratio. Allmice were immunised subcutaneously with 200 μg of bovine type IIcollagen in CFA. 21 days later, all mice were immunised subcutaneouslywith 100 μg of bovine type II collagen in IFA.

The mice started to develop signs and symptoms of arthritis followingthe ‘booster’ immunisation.

For macroscopic assessment of arthritis, the following signs weremonitored in each paw of each mouse three times per week and summed togenerate the Arthritic Index (AI) (the maximum AI for one animal is 16):

-   -   0=no visible effects of arthritis.    -   1=oedema and/or erythema of 1 digit.    -   2=oedema and/or erythema of 2 digits.    -   3=oedema and/or erythema of more than 2 digits.    -   4=severe arthritis of entire paw and digits.

Animals were sorted into treatment groups with a mean arthritic index of2.5 and then dosed once daily for 14 days with compound by oral gavagefor test compounds, or by subcutaneous injection at a dose of 10 mg/kgfor the positive control, etanercept. After completion of theexperiment, the mice were sacrificed.

The data were analysed by generating an average of the arthritic indexacross each treatment group. The mean arthritic index was then comparedto the arthritic index of control (untreated) animals using thefollowing formula to generate a percentage inhibition of disease.

${\%\mspace{14mu}{inhibition}\mspace{14mu}{of}\mspace{14mu}{disease}} = {100 - \left\lbrack {\frac{{average}\mspace{14mu}{arthritic}\mspace{14mu}{{index}:{{treated}\mspace{14mu}{animals}}}}{{average}\mspace{14mu}{arthritic}\mspace{14mu}{{index}:{{untreated}\mspace{14mu}{animals}}}}*100} \right\rbrack}$

The data summarised in the following table.

TABLE 8 Inhibition of Arthritis Dose % inhibition Compound (mg/kg/day)of disease ABD735 10 44 ABD899 10 77 HMC-C-01-A 10 40 HMC-C-01-A 3 60HMC-C-01-A 1 50 HMC-C-01-A 0.3 60 HMC-N-01-A 10 45 HMC-C-02-A 10 61HMC-N-02-A 10 36 HMC-C-01-B 10 26 HMC-N-01-B 10 → 1 (*) 38 (*) Reducedfrom 10 to 1 mg/kg/day due to mortality.

The data for several of the compounds are also illustrated in FIGS. 1and 2.

FIG. 1 shows six graphs, each of average arthritic index as a functionof time (dosing day) for test compound dosed at 10 mg/kg/day by oralgavage (open circles) and control (solid circles), for each of: (A)HMC-C-02-A (top left), (B) HMC-C-01-A (top middle), (C) HMC-N-02-A (topright), (D) HMC-N-01-A (bottom left), (E) HMC-C-01-B (bottom middle),(F) HMC-N-01-B (bottom right).

FIG. 2 shows two graphs, each of arthritic index as a function of time(dosing day) for test compound (open circles, open squares), control(solid circles) and positive control, the marketed drug etanercept(triangles), for each of (A) ABD899 at 10 mg/kg/day (left), (B)HMC-C-01-A at 0.3 mg/kg/day and 3 mg/kg/day (right).

These data indicate that the HMC compounds described herein showexcellent oral in vivo activity in preventing the progression ofestablished, severe arthritis.

Furthermore, the data show the exceptional activity of compoundHMC-C-01-A, which shows greater efficacy than the marketed treatment,etanercept, at low doses. This activity is particularly surprising sinceHMC-C-01-A is not the most active compound in either Biological Study 1or Biological Study 4. Additionally, the activity of HMC-C-01-A isgreater than that of the closely related compound, HMC-C-01-B, which ismore active than HMC-C-01-A in Biological Studies 1 and 4, showing thatdemonstrating that the identification of compounds with superiorefficacy is neither trivial nor predictable.

Biological Study 7 Maximum Tolerated Dose

The acute safety of the compounds was assessed in rats in order toenable determination of the maximum tolerated dose (MTD), an indicationof a compound's safety in animals. The higher the MTD, the greater isthe potential safety of the compound being tested.

Two male and two female Sprague-Dawley rats, aged 8-12 weeks, were dosedat each dose level in an ascending dose study design. The test compoundswere administered orally as a suspension formulated in 0.5%carboxymethylcellulose (CMC)/0.1% Tween-80 in a dose volume of 10 mL/kg.Animals were given free access to food throughout the study.

Animals were observed on at least two occasions during the first hourfollowing dosing, approximately 30 minutes apart, and thereafter athourly intervals for the remainder of the dosing day for at least 4hours. On subsequent days, the animals were observed at least once inthe morning and once towards the end of the day. The nature andseverity, where appropriate, of the clinical signs and time wererecorded at each observation. Observations included changes in the skin,fur, eyes, and mucous membranes, and also the respiratory, circulatory,autonomic, and central nervous system, as well as somatomotor activityand behaviour pattern. Particular attention was directed towards theobservation of tremors, convulsions, salivation, diarrhea, lethargy,sleep, and coma.

At the completion of the study, the results were expressed as themaximum tolerated dose for each compound, this being the highest of eachwithout unacceptable toxicity.

The maximum dose without unacceptable side-effects was determined to bethe MTD (expressed in mg/kg).

The data are summarised in the following table.

TABLE 9 Maximum Tolerated Dose Maximum tolerated dose (mg/kg) CompoundFemales Males ABD735 20 10 ABD899 30 30 REF001 7.5 10 HMC-C-03-A 30 30HMC-N-03-A 50 50 HMC-C-01-A ⁽¹⁾ 70 70 HMC-C-01-A ⁽²⁾ >500 >500HMC-N-01-A 80 80 HMC-C-02-A >200 >200 HMC-N-02-A >200 >200 ⁽¹⁾ Dosed toSprague Dawley rats as described. ⁽²⁾ Dosed to Han Wistar rats.

These data demonstrate that the HMC compounds described herein showgreatly increased safety as compared to the reference compounds ABD735,ABD899 and REF001.

The data also demonstrate that the improvement of acute safety isneither trivial nor predictable and that similar substitution patterns,for example those found in REF001 can lead to a decrease in acutesafety. Furthermore, the data show the exceptional safety of compoundsHMC-C-01A, HMC-C-02-A, HMC-N-01-A, and HMC-N-02-A, and particularly ofHMC-C-01-A.

The increases in the maximum tolerated dose seen with the HMC compoundsdescribed herein provide for an improved safety margin as compared tothe reference compounds and underline the potential of the HMC compoundsas oral drugs.

Biological Study 8 GreenScreen HC Genotoxicity and Cytotoxicity Assay

GreenScreen HC is a mammalian cell-based assay for measuring thegenotoxicity and cytotoxicity of chemical compounds and mixtures.

The term genotoxic is used to describe substances that are capable ofcausing damage to genetic material (DNA) within a cell, which mayultimately have mutagenic, carcinogenic or teratogenic effects inhumans.

The GreenScreen assay reports genotoxic stress as an increase influorescence from a genetically modified derivative of the p53-competentTK6 human lymphoblastoid cell line. In the modified cells, regulatoryDNA sequences which promote the transcription of the GADD45a genecontrol the expression of Green Fluorescent Protein (GFP). GADD45a has acentral role in genomic integrity, and genotoxic stress induces itstranscription. Exposure to a genotoxic compound therefore increases theexpression of GFP, which is observed as an increase in fluorescence andwhich is monitored by fluorescent detection using a plate reader or flowcytometer. In the GreenScreen HC assay fluorescence is normalised to theoptical absorbance measurement to correct for variation in cell yieldcaused by loss of cell viability or cytotoxicity. The statisticallydefined threshold for a positive result in the GreenScreen HC assay is1.5, i.e., 50% induction over and above the baseline for thevehicle-treated control.

Cytotoxicity is measured in the GreenScreen assay alongside genotoxicityand the results of the cytotoxicity assessment are used to normalise thegenotoxicity measurements as described above.

In the GreenScreen HC assay, cell viability is assessed using apropidium iodide uptake assay. Propidium iodide is an intercalatingagent and a fluorescent molecule that can be used to stain cells. It iscommonly used to quantitatively assess DNA content to evaluate cellviability or DNA content in cell cycle analysis and can be used todifferentiate necrotic, apoptotic and normal cells (see, e.g., Dengleret al., 1995, Anticancer Drugs, Vol. 6, No. 4, pp. 522-532). Exposure toa cytotoxic compound increases the uptake of propidium iodide, which ismonitored by optical absorbance and is proportional to cellproliferation. Cytotoxic compounds are those that show an observeddecrease in relative population survival below a significance thresholdset at 90% compared to vehicle-treated control, at one or more testconcentrations.

A dilution series of each test compound is generated in parallel in a96-well, black microplate with an optically clear base. A standardgenotoxic compound (methyl methanesulfonate, MMS) is added as anintra-plate quality control check. The plates are analysed at 24 hourand 48 hour time points using a microplate reader, which providesmeasurements of light absorbance and fluorescence for cells andsolutions in the microplates wells.

In addition to measurements of cell viability and genotoxicity describedabove, the GreenScreen assay incorporates an assessment of the effectsof metabolic activation in the assay, the “GreenScreen HC S9 assay”. Toaccomplish this, the cells are incubated both in the presence of andabsence of the post-mitochondrial supernatant liver extract (known as“S9”). S9 is routinely used in genetic toxicology to supplement the testcells with mammalian phase I metabolism. To assess metabolic activation,compounds are incubated with the TK6 strain in the presence of 1% v/v S9fraction mix for 3 hours with a subsequent 45 hour recovery time. Afterthe recovery period, the GFP fluorescence signal and cell viability(assessed by propidium iodide uptake) are measured using a flowcytometer. Cyclophosphamide, a commonly used control in genotoxicitystudies utilising metabolic activation, is used as the positive controlin the assay. In the GreenScreen HC S9 assay, genotoxicity is evaluatedby induction in GFP expression quantified using the mean samplefluorescence and cytotoxicity using the propidium iodide uptake assaydescribed above. The statistically defined threshold for a positivegenotoxicity result is 1.3, i.e. 30% induction over and above thebaseline for the vehicle-treated control and the result is reported aspositive or negative. Cytotoxic compounds are those that show anobserved decrease in relative population survival below a significancethreshold set at 90% compared to vehicle-treated control, at one or moretest concentrations.

Stably transfected reporter cell lines were derived from thep53-competent human lymphoblastoid TK6 cell line. The reporter cell linecarries an episomally replicating plasmid bearing the upstream promoterregion and other regulatory sequences of the human GADD45a gene linkedto a human optimised Green Fluorescent Protein (GFP) gene. The controlcell line carries an identical plasmid except for the removal of 4 basepairs at the start of the EGFP gene, such that GFP is not produced. Bothplasmids also carry a gene conferring resistance to hygromycin B to thecell line, allowing continued selection for plasmid presence (200 μg/mLhygromycin B; Invitrogen Corporation, Carlsbad, Calif.). Both cell lineswere maintained in complete culture medium (RPMI 1640 with GlutaMAX™ and25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (InvitrogenCorporation) supplemented with 10% (v/v) heat-inactivated donor horseserum (Lonza Wokingham Ltd, Wokingham, UK), 10 mM sodium pyruvate(Invitrogen Corporation) and 5000 U/ml penicillin G sodium with 5000μg/ml streptomycin sulphate (Invitrogen Corporation)) at 37° C. with 5%CO₂ in a humidified atmosphere.

Each assay plate is prepared to assess each test compounds across nineserial 2-fold dilutions, with both the reporter strain and a controlcell line which contains the same genetic modifications as the teststrain, but does not produce GFP because of a minor change to thesequence at the beginning of the inserted GFP gene. Forty-eight hourexposures were performed in sterile black walled, optically clear,flat-bottomed polystyrene 96-well microplates. Duplicate wells of both‘high’ (50 μg/mL) and ‘low’ (10 μg/mL) concentrations of methylmethanesulfonate were included as intra-plate positive controls toprovide data acceptance. Other control wells included vehicle-treated(1% v/v DMSO) cells as a negative control, assay medium alone to checkfor contamination, and test compound with assay medium to determine anyinherent fluorescence and/or optical absorbance due to colour orprecipitation.

Breathable membranes (BreathEasy, Diversified Biotech, USA) were appliedto the microplates prior to their vigorous shaking (20 seconds). Thiswas followed by static incubation for 48 hours at 37° C. and 5% CO₂ in ahumidified atmosphere. After 48 hours, the microplates were vigorouslyshaken for 10 seconds to resuspend cells prior to spectrophotometricdata acquisition. Fluorescence data were normalized to absorbance datato give a ‘brightness’ value relative to the average of vehicle-treatedcontrols and plotted against compound concentration. A brightnessincrease of ≧50% was classified as a positive result for genotoxicity:this reflects an increase of greater than three times the standarddeviation in fluorescence data from vehicle-treated andnon-genotoxin-treated cells. The absorbance data were normalized to thevehicle-treated control and plotted as cytotoxicity. Cytotoxicity of 90%reflects a statistically significant fall in cell yield and is recordedas a growth inhibitory toxic effect.

Raw data collected from GreenScreen HC assay microplates, using themicroplate were automatically analysed using a GreenScreen HC softwaretemplate to produce a result summary for the following parameters:lowest effective concentrations (LECs) that cause positive results inthe propidium iodide exclusion assay (i.e., loss of cell viability) at24 hours and 48 hours in the presence and absence of S9 fraction;negative or positive effects on genotoxicity.

The results are summarised in the following table.

TABLE 10 Cytotoxicity and Genotoxicity Cytotoxicity, LEC (μM) Compound24 h 48 h, −S9 48 h, +S9 Genotoxicity ABD735 0.31 0.31 5 Negative ABD8360.04 0.01 40 Positive ABD899 ⁽¹⁾ <0.001 <0.001 0.32 Positive ABD899 ⁽²⁾0.64 0.08 81.9 Negative ABD900 0.29 0.07 74.1 Negative REF001 0.0390.039 0.63 Positive HMC-C-01-A 0.47 0.12 60 Negative HMC-C-02-A >1000 60120 Negative HMC-C-03-A 0.24 0.06 15 Negative HMC-N-01-A 0.94 0.12 60Negative HMC-N-02-A 0.94 0.47 120 Negative HMC-N-03-A 0.94 0.24 30Negative HMC-N-04-A 0.47 0.12 30 Negative HMC-C-01-B 0.08 0.005 4.82Negative HMC-C-03-B 0.17 0.08 10.8 Positive HMC-N-01-B 0.01 0.05 5.85Negative HMC-N-02-B 0.04 0.01 10.2 Negative ⁽¹⁾ A lab scale batch wastested in the range 1.3 mM to 0.3 pM. ⁽²⁾ A mid scale batch was testedin the range 164 μM to 0.003 μM.

These data indicate that the HMC compounds described herein showenhanced or at least similar cytotoxicity and genotoxicity to thereference compounds and are markedly better than ABD836 and REF001 inparticular.

The data also demonstrate that the changes required to improve thecytotoxicity or genotoxicity profile is neither trivial nor predictable.This is particularly highlighted by the profiles of the closely relatedcompounds ABD836 and HMC-N-02-A.

Furthermore, the data show the exceptional safety with respect togeneral cytotoxicity of compounds HMC-C-02-A, HMC-C-01-A, HMC-N-02-A,and HMC-N-01-A.

Biological Study 9 hERG Ion Channel Assay

Inhibition of the human Ether-à-go-go-Related Gene (hERG) ion channelmediates the repolarizing IKr current in the cardiac action potential,thereby indicating that it contributes to the electrical activity thatcoordinates the beating of the heart. When the ability of hERG toconduct electrical current across the cell membrane is inhibited orcompromised it can result in a potentially fatal disorder called long QTsyndrome. This association between hERG and long QT syndrome has madehERG inhibition an important anti-target that must be avoided duringdrug development.

The activity of the compounds against the hERG ion channel was tested.The assay was conducted using the automated path-clamp, Q-patch methodusing stably transfected Chinese Hamster Ovary cells (hERG-CHO).hERG-CHO cells were cultured in F-12 Kaighn's Nutrient Mixture medium(Invitrogen)+10% FBS at 37° C. for 1-3 days. Cells were kept at 30° C.for 24 to 48 hours prior to patch clamping in order to increase the hERGcurrent amplitude. Subsequently, the cells were harvested bytrypsinisation, and kept in Serum Free Medium (SFM) in the Q-patch cellpreparation state for up to 6 hours at room temperature before beingwashed and re-suspended in extracellular solution and applied to thepatch clamp sites for data recording.

Patch-clamp voltage protocol: After whole cell configuration wasachieved, the cell was held at −80 mV. A 50 millisecond pulse to −40 mVwas delivered to measure the leaking current, which was subtracted fromthe tail current on-line. Then the cell was depolarized to +20 mV for 2seconds, followed by a one second pulse to −40 mV to reveal hERG tailcurrent. This paradigm was delivered once every 5 seconds to monitor thecurrent amplitude.

Extracellular solution: 137 mM NaCl, 4 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂,10 mM D(+)-glucose, 10 mM HePES buffer (pH adjusted to 7.4 with NaOH).

After the whole cell configuration was achieved, the extracellularsolution (control) was applied first and the cell was stabilized for 2minutes in extracellular solution. The test compound was then appliedfrom low concentrations to high concentrations cumulatively. The cellwas incubated with each test concentration for 5 minutes. During eachincubation, the cell was repetitively stimulated using the voltageprotocol described above, and the tail current amplitude wascontinuously monitored.

Acceptance Criteria:

(1) Peak tail current >100 pA in control.

(2) Initial run-down <30% and the run-down stops before firstapplication of the test compound.

(3) Leak currents <50% of the control peak tail currents at any time.

(4) rs<20 M4 throughout the experiment.

The degree of inhibition (%) was obtained by measuring the tail currentamplitude, induced by a one second test pulse to −40 mV after a twosecond pulse to +20 mV, before and after incubation with the testcompound. The difference in current was normalized to control andmultiplied by 100 in order to obtain the percent inhibition.

Concentration (log) response curves were fitted to a logistic equation(three parameters assuming complete block of the current at very hightest compound concentrations) to generate estimates of the 50%inhibitory concentration (IC₅₀). The concentration-response relationshipof each compound was constructed from the percentage reductions ofcurrent amplitude by sequential concentrations.

The results are summarised in the following table.

TABLE 11 hERG Ion Channel Inhibition % inhibition Compound IC₅₀ (μM) ⁽¹⁾@ 30 μM ABD599 4.9 85 ABD735 —  62.5 ABD836 — 50 ABD899 2.9  100 ⁽²⁾ABD899  53 ⁽³⁾ ABD900 51 REF001 — 60 HMC-C-01-A 19.3 79 HMC-C-02-A 25 57HMC-C-03-A >30 45 HMC-C-04-A 69 HMC-N-01-A 183  2 HMC-N-02-A 231 31HMC-N-03-A >30 13 HMC-N-04-A >30 26 HMC-C-01-B 74 HMC-C-02-B 94HMC-C-03-B 36 HMC-N-01-B 27 HMC-N-02-B 23 HMC-N-03-B 47 HMC-N-04-B 60⁽¹⁾ IC50s were calculated using a four parameter logistic equationcalculated automatically in Grafit version 6.0.12 (Erithacus SoftwareLtd., by Dr Robin Leatherbarrow). ⁽²⁾ A lab-scale batch was tested. ⁽³⁾A mid-scale batch was tested.

The data demonstrate that the HMC compounds described herein havecardiac safety properties required for an orally active drug, and havesafety advantages as compared to the reference compounds, such as ABD599and ABD899, with HMC-N-01-A, HMC-N-02-A and HMC-N-03-A showing aparticularly positive profile.

Biological Study 10 Human Primary Leucocyte Studies

The human immune system involves a complex set of cells and organs thatcan be adversely affected by drugs or chemicals. This results inincreased susceptibility to infections, tumours, allergic responses,autoimmune reactions or other forms of immune system diseases. Theassessment of immunotoxicity is, therefore, an important component ofsafety evaluation of new pharmaceuticals, and the ideal profile for apotential anti-inflammatory drug that acts via modulating the immunesystem is one that demonstrates selectively for certain subsets ofimmune-system cells with no effects on others, thereby avoiding generalimmune activation or suppression. For example, a drug that reduces theviability or activity of monocytes with no effects on neutrophils wouldbe expected to possess anti-inflammatory properties with application ina number of diseases without compromising the ability of a patient torespond to, and clear, infections.

The ability of a reference compound (ABD735) to influence the viabilityof human white blood cells was investigated using cells derived fromhuman whole blood. The effects of a reference compound (ABD735) weretested on the following human white blood cell types: neutrophils,monocytes, B-lymphocytes, and T-lymphocytes, comprising both the CD4positive (T-helper) and CD8-positive (T-cytotoxic) populations. Each ofthese cell types has a different function in the human immune system.

Neutrophils are also known as granulocytes. They are the most abundantwhite blood cells in man and form part of the innate immune system.Neutrophils act as the primary response to the acute phase ofinflammation caused by bacteria infection.

Monocytes are part of the innate immune system found in the circulation.Peripheral blood monocytes differentiate to form macrophages, which arethe precursors of a number of cells involved in modulating the immuneresponse, including dendritic cells. The primary function ofmonocyte-macrophages is to act as immune effector cells. They areheavily involved in the pathogenesis of several chronic inflammatoryconditions including rheumatoid arthritis and multiple sclerosis.

T-lymphocytes are part of the adaptive immune system and play a centralrole in immunity. There are several subsets of T-lymphocytes, each ofwhich possess a different function in the immune system. T-cytotoxiclymphocytes are also known as CD8+ T-cells due to the presence of amolecule called CD8 on their surface. Their role is to destroy infectedcells and tumour cells, but under inflammatory conditions, they can alsoact to exacerbate disease. T-helper lymphocytes are also known as CD4+T-cells due to the presence of a molecule called CD4 on their surface.The function of T-helper cells is to assist in maturing B-lymphocytesand to activate cytotoxic T-cells.

B-lymphocytes are part of the adaptive immune system. Their principalfunctions are to make antibodies and act as antigen-presenting cells,which allow T-lymphocytes to recognise foreign antigens.

The effects of the reference compound (ABD735) on the viability ofneutrophils, monocytes, CD4+ and CD8+ T-cells and B-lymphocytes wasassessed under both resting and stimulated conditions. Restingconditions were used to assess likely effects on normal cells found inhuman blood, and stimulated conditions were used to assess the effectsof the compound under disease conditions.

Effects on cell viability were assessed using flow cytometry. Flowcytometry is a laser-based, biophysical technology employed in cellcounting, cell sorting, and biomarker detection, by suspending cells ina stream of fluid and passing them by an electronic detection apparatus.It allows simultaneous multi-parametric analysis of the physical andchemical characteristics of up to thousands of particles per second. Thesorting of cells using flow cytometry requires the use of specific cellmarkers. To quantify cell numbers, counting beads are used as aninternal standard. Counting beads are a calibrated suspension ofmicrobeads which are added to a known volume of sample so the samplevolume per bead is known. This allows the absolute determination of cellnumber in a sample. In addition, for B- and T-lymphocytes, cellproliferation was measured using a dye known as Cell Proliferation Dye(eFluor® 450). eFluor® 450 is an organic dye which can be conjugated tocell-specific markers. It fluoresces when excited by a laser in a mannerproportional to the number of cells bound by the conjugate. Its signaltherefore provides an indication of the proliferation of specific celltypes.

Neutrophil Isolation and Assessment of Impact on Survival:

Neutrophils were isolated from whole blood (collected from healthydonors) by two-step density gradient Histopaque® 1077 and Histopaque®1119. Polymorphonuclear cells were washed in RPMI media supplementedwith antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL) andresuspended at 2×10⁶ cells/mL in RPMI supplemented with 10% foetal calfserum (FCS) and antibiotics (penicillin 100 U/mL, streptomycin 100μg/mL). Then 50 μL of the cell suspension was added to a 96 well plate,with or without reference compound (ABD735) (at 30, 10, 3, 1, or 0.3 μM)or control (0.3% DMSO), with or without dexamethasone (1 μM) andincubated at 37° C./5% CO₂ for 24 hours.

Following incubation, the cells were fixed and an exact volume ofcounting beads was added to each tube to calculate the number of livecells.

Monocyte Isolation and Assessment of Impact on Survival:

PBMCs (peripheral blood mononuclear cells) were collected from healthydonors (all male) and isolated by centrifugation on a layer of FicollPaque (GE Healthcare, UK). Cells were then resuspended (3×10⁶ cells/mL)in RPMI supplemented with 10% FCS and antibiotics (penicillin 100 U/mL,streptomycin 100 μg/mL) and 1 mL of cell suspension was added to a48-well plate, which was then incubated for 1 hour at 37° C./5% CO₂.Subsequently, the supernatant was aspirated to remove non-adherentcells, which was followed by two washing steps to ensure removal ofnon-adhered cells. Adhered cells (monocytes) were then stimulated witheither LPS (10 ng/mL), TNFα (10 ng/mL) or macrophage colony stimulatingfactor (M-CSF) (10 ng/mL) in the absence (control) or presence ofreference compound (ABD735) (at 30, 10, 3, 1, or 0.3 μM) for 48 hours at37° C./5% CO₂. Subsequently, the plates were centrifuged and themonocytes washed in PBS before addition of PBS plus EDTA (10 mM) andincubation at 4° C. for 20 minutes to help detach the cells, which wasfacilitated by pipetting. The monocytes were added to Falcon tubes andwashed by centrifugation with PBS before an exact volume of countingbeads was added to each tube to calculate the number of live cells.

B-Lymphocyte Isolation and Assessment of Impact on Survival:

PBMCs (peripheral blood mononuclear cells) were collected from healthydonors (all female) and isolated by centrifugation on a layer of FicollPaque (GE Healthcare, UK). The cells were then resuspended in RPMIsupplemented with 10% FCS and antibiotics (penicillin 100 U/mL,streptomycin 100 μg/mL). Cell suspension was added to a 96-well platewith or without reference compound (ABD735) (at 30, 10, 3, 1, or 0.3 μM)or control (0.3% DMSO) with or without a combination of Staphylococcusaureus Cowan I (SAC) and interleukin-2 (SAC/IL-2) (1:10,000 and 2 ng/mL)and incubated at 37° C./5% CO₂ for 48 hours. Following incubation, thecells were stained with anti-human CD19 before an exact volume ofcounting beads was added to each tube to calculate the number of livecells.

B-Cell Isolation and Assessment of Impact on Proliferation:

PBMCs (peripheral blood mononuclear cells) were collected from healthydonors (all female) and isolated by centrifugation on a layer of FicollPaque (GE Healthcare, UK).

The cells were then stained with Cell Proliferation Dye, which measuresthe proliferation of cells (eFluor® 450) and re-suspended in RPMIsupplemented with 10% FCS and antibiotics (penicillin 100 U/mL,streptomycin 100 μg/mL). Cell suspension was added to a 96 well platewith or without reference compound (ABD735) (at 30, 10, 3, 1, or 0.3 μM)or control (0.3% DMSO) with or without a combination of Staphylococcusaureus Cowan I (SAC) and interleukin-2 (SAC/IL-2) (1:10,000 and 2 ng/mL)and incubated at 37° C./5% CO₂ for 5 days. Following incubation, thecells were stained with anti-human CD19, a marker of B-lymphocytes,before being analysed on a flow cytometer. Cells that were e450 low weredeemed to have proliferated.

T-Cell Isolation and Assessment of Impact on Survival:

PBMCs (peripheral blood mononuclear cells) were collected from healthydonors (all female) and isolated by centrifugation on a layer of FicollPaque (GE Healthcare, UK). The cells were then resuspended to 2×10⁶cells/mL in RPMI supplemented with 10% FCS and antibiotics (penicillin100 U/mL, streptomycin 100 μg/mL). Cell suspension was added to a96-well plate with or without reference compound (ABD735) (at 30, 10, 3,1, or 0.3 μM) or control (0.3% DMSO) with or without phytohaemagglutinin(PHA, 5 μg/mL) and incubated at 37° C./5% CO₂ for 48 hours. Followingincubation, the cells were stained with anti-human CD8 or CD4 before anexact volume of counting beads was added to each tube to calculate thenumber of live cells.

T-Cell Isolation and Assessment of Impact on Proliferation:

PBMCs (peripheral blood mononuclear cells) were collected from healthydonors (all female) and isolated by centrifugation on a layer of FicollPaque (GE Healthcare, UK).

The cells were then stained with Cell Proliferation Dye (eFluor® 450)and re-suspended in RPMI supplemented with 10% FCS and antibiotics(penicillin 100 U/mL, streptomycin 100 μg/mL). Cell suspension was addedto a 96-well plate with or without reference compound (ABD735) (at 30,10, 3, 1, or 0.3 μM) or control (0.3% DMSO) with or withoutphytohaemagglutinin (PHA, 5 μg/mL) and incubated at 37° C./5% CO₂ for 5days. Following incubation, the cells were stained with eitheranti-human CD8 or CD4 before being analysed on a flow cytometer. Cellsthat were e450 low were deemed to have proliferated.

The average results (n=3) for the reference compound (ABD735) wereexpressed as a fold change versus the average control value. The datawere then plotted graphically using software from Grafit (ErithacusSoftware). Each experiment was repeated three times and the data arepresented as the mean from all experiments.

The results are summarised in the following tables.

TABLE 12 Cell Viability in the presence of ABD735 (3 μM) Fold changeCell type Stimulation in viability Neutrophils — 1.0 Dexamethasone 0.8B-lymphocytes — 0.9 SAC/IL-2 1.1 CD4₊ T-lymphocytes — 0.9phytohaemagglutinin 0.6 CD8₊ T-lymphocytes — 0.8 phytohaemagglutinin 0.7Monocytes — 0.7 M-CSF 1.1 LPS 1.0 TNFα 0.5

TABLE 13 Cell Proliferation in the presence of ABD735 (3 μM) Fold changeCell type Stimulation in proliferation B-lymphocytes — 0.9 SAC/IL-2 0.6CD4₊ T-lymphocytes — 1 phytohaemagglutinin 0.6 CD8₊ T-lymphocytes — 0.7phytohaemagglutinin 0.3

The results from a single high concentration (3 μM) of the referencecompound (ABD735) across the leucocyte types are shown above. In thisassay, a fold-change of 0.5 or lower is significant for loss ofviability, and a fold-change of 0.6 or lower is significant forreductions in proliferation.

It can be seen from these data that the reference compound (ABD735)selectively reduces the viability of monocytes in the presence of TNFαonly, and not in the presence of either M-CSF or LPS. In addition, thereference compound (ABD735) has very little effect on the viability ofthe other leucocyte populations indicating that compounds from thisseries are not generally immunosuppressive. The reference compound(ABD735) is also shown to reduce the proliferation of lymphocytes, withthe most profound effects seen in stimulated CD8+T-lymphocytes. Aprofile such as this, with selective reduction in the proliferation oflymphocyte subpopulations is an attractive mechanistic profile for thetreatment of diseases of inflammation such as rheumatoid arthritis,systemic lupus erythematosus, Crohn's disease, and multiple sclerosis,as well as for the treatment of leukaemias including: T-cell lymphoma,such as extranodal T-cell lymphoma, cutaneous T-cell lymphoma,anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma;B-cell lymphoma, such as Hodgkin's and non-Hodgkins lymphomas, includingdiffuse large B-cell lymphoma, follicular lymphoma, mucosa-associatedlymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle celllymphoma, and Burkitt's lymphoma; and other leukaemias, such as chroniclymphocytic leukaemia and multiple myeloma.

Biological Study 11 Rat Pristane-Induced Arthritis Study

Female Lewis rats were used for all procedures. Animals were housed ingroups of 5, and were maintained at 21° C.±2° C. on a 12-hour light/darkcycle with food and water ad libitum. Arthritis was induced by theadministration of 0.3 mL pristane intra-dermally at the base of thetail. The rats started to develop signs and symptoms of arthritisapproximately seven days after the pristane injection.

For macroscopic assessment of arthritis, the following signs weremonitored in each paw of each mouse three times per week and summed togenerate the Arthritic Index (AI) (the maximum AI for one animal is 16):

-   -   0=no visible effects of arthritis.    -   1=oedema and/or erythema of 1 digit.    -   2=oedema and/or erythema of 2 digits.    -   3=oedema and/or erythema of more than 2 digits.    -   4=severe arthritis of entire paw and digits including limb        deformation and ankylosis of the joint.

Animals were sorted into treatment groups prior to pristaneadministration and dosed once daily for 28 days with test compound atdoses of 3 and 10 mg/kg/day by oral gavage, or by intraperitonealinjection at a dose of 0.05 mg/kg for the positive control,methotrexate. After completion of the experiment, the rats weresacrificed.

The data were analysed by generating an average of the arthritic indexacross each treatment group. The mean arthritic index was then comparedto the arthritic index of control (untreated) animals using thefollowing formula to generate a percentage inhibition of disease.

${\%\mspace{14mu}{inhibition}\mspace{14mu}{of}\mspace{14mu}{disease}} = {100 - \left\lbrack {\frac{{average}\mspace{14mu}{arthritic}\mspace{14mu}{{index}:{{treated}\mspace{14mu}{animals}}}}{{average}\mspace{14mu}{arthritic}\mspace{14mu}{{index}:{{untreated}\mspace{14mu}{animals}}}}*100} \right\rbrack}$

The data summarised in the following table.

TABLE 14 Inhibition of Arthritis Dose % inhibition Compound (mg/kg/day)of disease ABD899 3 21 ABD899 10 ND⁽¹⁾ HMC-C-01-A 3 40 HMC-C-01-A 10 67HMC-N-01-A 3 39 HMC-N-01-A 10 60 ⁽¹⁾Dosing was terminated on day 10 dueto adverse effects.

The data for several of the compounds are also illustrated in FIG. 3.

FIG. 3 shows six graphs, each of average arthritic index as a functionof time (dosing day) for test compound (open circles), control (solidcircles), and positive control, methotrexate (triangles) for each of:(A) ABD899 dosed at 3 mg/kg/day (top left), (B) HMC-C-01-A dosed at 3mg/kg/day (top middle), (C) HMC-N-01-A dosed at 3 mg/kg/day (top right),(D) ABD899 dosed at 10 mg/kg/day (bottom left), (E) HMC-C-01-A dosed at10 mg/kg/day (bottom middle), and (F) HMC-N-01-A dosed at 10 mg/kg/day(bottom right).

These data indicate that the HMC compounds described herein showexcellent oral in vivo activity in preventing the progression ofpristane induced arthritis, whilst compound ABD899 had limited effect ondisease in this model, in spite of good activity in Biological Study 6.In addition, compounds HMC-C-01-A and HMC-N-01-A were tolerated well bythe animals during extended dosing, whilst ABD899 was poorly toleratedsuch that it was necessary to terminate dosing and remove the animalsreceiving it from the study (shown in FIG. 3, panel D). Furthermore,compound HMC-C-01-A showed particularly good efficacy in the model,which was equivalent to that of the marketed first-line therapy forrheumatoid arthritis, methotrexate.

The data further show that the identification of compounds with superiorefficacy is neither trivial nor predictable.

The foregoing has described the principles, preferred embodiments, andmodes of operation of the present invention. However, the inventionshould not be construed as limited to the particular embodimentsdiscussed. Instead, the above-described embodiments should be regardedas illustrative rather than restrictive. It should be appreciated thatvariations may be made in those embodiments by workers skilled in theart without departing from the scope of the present invention.

REFERENCES

A number of publications are cited herein in order to more fullydescribe and disclose the invention and the state of the art to whichthe invention pertains. Full citations for these publications areprovided below. Each of these publications is incorporated herein byreference in its entirety into the present disclosure, to the sameextent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

-   Akahoshi et al., 2008, “Promoter polymorphisms in the IRF3 gene    confer protection against systemic lupus erythematosus”, Lupus, Vol.    17 pp. 568-574.-   Astry et al., 2011, “A cytokine-centric view of the pathogenesis and    treatment of autoimmune arthritis”, J Interferon Cytokine Res., Vol.    31, pp. 927-940.-   Bahmanyar et al., 2010, “Aminotriazolopyridines and their use as    kinase inhibitors”, international patent publication number WO    2010/027500 A1 published 11 Mar. 2010.-   Baud et al., 1999, “Signalling by proinflammatory cytokines:    oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK    activation and target gene induction via an amino-terminal effector    domain”, Genes Dev., Vol. 13, pp. 1297-1308.-   Baud et al., 2009, “Is NFκB a good target for cancer therapy? Hopes    and pitfalls”, Nat. Rev. Drug Disc., Vol. 8, pp. 33-40.-   Billiau, 2010, “Etanercept improves linear growth and bone mass    acquisition in MTX-resistant polyarticular-course juvenile    idiopathic arthritis”, Rheumatology (Oxford), Vol. 49, pp.    1550-1558.-   Bladh et al., 2006, “Novel sulphonamide derivatives as    glucocorticoid receptor modulators for the treatment of inflammatory    diseases”, international patent publication number WO 2006/046916 A1    published 4 May 2006.-   Brennan et al., 1992, “Enhanced expression of tumor necrosis factor    receptor mRNA and protein in mononuclear cells isolated from    rheumatoid arthritis synovial joints”, Eur. J. Immunol., Vol. 22,    pp. 1907-1912.-   Brennan et al., 1996, “Cytokines in autoimmunity”, Curr. Opin.    Immunol., Vol. 8, pp. 872-877.-   Chen et al., 2012, “High-affinity and selective dopamine D₃ receptor    full agonists”, Bioorg. & Med. Chem. Lett., Vol. 22, pp. 5612-5617.-   Childs, L. M., et al., 2001, “Efficacy of etanercept for wear    debris-induced osteolysis”, Journal of Bone and Mineral Research,    Vol. 16, No. 2, pp. 338-347.-   Dallas et al., 2011, “Osteoimmunology at the nexus of arthritis,    osteoporosis, cancer, and infection”, J. Clin. Invest., Vol. 121,    pp. 2534-2542.-   Dimitris et al., 1998, “The Pathophysiologic Roles of Interleukin-6    in Human Disease”, Ann Intern Med., Vol. 128, No. 2, pp. 127-137.-   Dvorak et al., 1996, “Comparative ultrastructural morphology of    human basophils stimulated to release histamine by anti-IgE,    recombinant IgE-dependent histamine-releasing factor, or monocyte    chemotactic protein-1”, Journal of Allergy and Clinical Immunology,    Vol. 98, pp. 355-370.-   Feldmann et al., 1994, “TNF alpha as a therapeutic target in    rheumatoid arthritis,” Circ. Shock, Vol. 43, pp. 179-184.-   Feldmann et al., 1996, “Rheumatoid arthritis”, Cell, Vol. 85, pp.    307-310.-   Feldmann et al., 2001, “The role of TNF alpha and IL-1 in rheumatoid    arthritis,” Curr. Dir. Autoimmun., Vol. 3, pp. 188-199.-   Firestein, 2005 “Immunologic mechanisms in the pathogenesis of    rheumatoid arthritis”, J. Clin. Rheumatol., Vol. 11. pp. S39-S44.-   Fu et al., 2011, “Association of a functional IRF7 variant with    systemic lupus erythematosus”, Arthritis Rheum., Vol. 63, pp.    749-754.-   Gabay, 2006, “Interleukin-6 and chronic inflammation”, Arthritis    Research & Therapy, Vol. 8 (Suppl 2), S3.-   Galli et al., 1989, “IgE, Mast Cells and the Allergic Response”,    Ciba Foundation Symposium, Vol. 147, pp. 53-73.-   Gottlieb, 2005, “Psoriasis: Emerging Therapeutic Strategies”, Nat.    Rev. Drug Disc., Vol. 4, pp. 19-34.-   Greig et al., 2006, “Development and characterization of    biphenylsulfonamides as novel inhibitors of bone resorption”, J.    Med. Chem., Vol 49: pp 7487-7492.-   Greig et al., 2008, “Biphenyl-4-yl-sulfonic acid arylamides and    their use as therapeutic agents”, international patent publication    number WO 2008/114022 A1 published September 2008.-   Greig et al., 2010a, “Aryl-phenyl-sulfonamido-cycloalkyl compounds    and their use”, international patent publication number WO    2010/032009 A1 published March 2010.-   Greig et al., 2010b, “Aryl-phenyl-sulfonamido-phenylene compounds    and their use”, international patent publication number WO    2010/032010 A1 published March 2010.-   Greig et al., 2013, “Development of triarylsulfonamides as novel    anti-inflammatory agents”, Bioorq. & Med. Chem. Lett., Vol. 23, pp.    816-820.-   Hadida et al., 2007, “Heterocyclic modulators of ATP-binding    cassette transporters”, international patent publication number WO    2007/056341 A1 published 18 May 2007.-   Hu et al., 2011, “A meta-analysis of the association of IRF5    polymorphism with systemic lupus erythematosus International”,    Journal of Immunogenetics, Vol. 38, pp. 411-417.-   Jimi et al., 2004, “Selective inhibition of NF-kappa B blocks    osteoclastogenesis and prevents inflammatory bone destruction in    vivo”, Nat. Med., Vol. 10, pp. 617-624.-   Joosten et al., 1996, “Anticytokine treatment of established type II    collagen-induced arthritis in DBA/1 mice. A comparative study using    anti-TNF alpha, anti-IL-1 alpha/beta, and IL-1Ra,” Arthritis Rheum.,    Vol. 39, pp. 797-809.-   Karsenty et al., 2002, “Reaching a genetic and molecular    understanding of skeletal development”, Dev. Cell., Vol. 2, pp.    389-406.-   Klareskog et al., 2006, “Mechanisms of disease: Genetic    susceptibility and environmental triggers in the development of    rheumatoid arthritis,” Nat. Clin. Pract. Rheumatol., Vol. 2, pp.    425-433.-   Korzenik et al., 2006, “Evolving knowledge and therapy of    inflammatory bowel disease,” Nat. Rev. Drug Disc., Vol. 5, pp.    197-209.-   Krausgruber et al., 2011, “IRF5 promotes inflammatory macrophage    polarization and TH1-TH17 responses”, Nat. Immunol., Vol. 12, pp.    231-238.-   Li et al., 2008, “A tumor necrosis factor-α-mediated pathway    promoting autosomal dominant polycystic kidney disease”, Nature    Medicine, Vol. 14, No. 8, pp. 863-868.-   Liu, 2005, “Molecular mechanism of TNF signalling and beyond,” Cell    Res., Vol. 15, pp. 24-27.-   Long, 2012, “Osteoimmunology: the expanding role of immunoreceptors    in osteoclasts and bone remodeling”, Bone Key Rep., Vol. 1, p. 59.-   Malemud et al., 2010, “Myeloid-related protein activity in    Rheumatoid Arthritis”, International Journal of Interferon, Cytokine    and Mediator Research, Vol. 2, pp. 97-111.-   Mantovani, 2009, “Inflaming metastasis”, Nature, Vol. 457, pp.    36-37.-   Mazzucchelli et al., 1996, “Differential in situ expression of the    genes encoding the chemokines MCP-1 and RANTES in human inflammatory    bowel disease”, J. Pathol., Vol. 178, No. 2, pp. 201-206.-   McInnes et al., 2007, “Cytokines in the pathogenesis of rheumatoid    arthritis”, Nat. Rev. Immunol., Vol. 7, pp. 429-442.-   Minamino et al., 2012, “IRF-2 regulates B-cell proliferation and    antibody production through distinct mechanisms”, Int Immunol., Vol.    24, pp. 573-581.-   Mount et al., 2005, “Rheumatoid arthritis market”, Nat. Rev. Drug    Disc., Vol. 2, pp. 11-12.-   O'Shea et al., 2013, “Janus kinase inhibitors in autoimmune    diseases”, Annals of Rheumatic Disease, Vol. 72, Supplement 2, pp.    111-115.-   Ogata et al., 2012, “Safety and Efficacy of Tocilizumab for the    Treatment of Rheumatoid Arthritis”, Clin Med Insights Arthritis    Musculoskelet Disord., Vol. 5, pp. 27-42.-   Parameswaran et al., 2010, “Tumor necrosis factor-α signaling in    macrophages”, Crit. Rev. Eukaryot. Gene Expr., Vol. 20, pp. 87-103.-   Philchenkov et al., 2004, “Caspases and cancer: mechanisms of    inactivation and new treatment modalities”, Exp. Oncol., Vol 26, pp    82-97.-   Pisetsky, 2012, “Advances in the treatment of inflammatory    arthritis”, Best Pract. Res. Clin. Rheumatol., Vol. 26. pp. 251-261.-   Ralston et al., 2005, “Aryl alkyl sulfonamides as therapeutic agents    for the treatment of bone conditions”, international patent    publication number WO 2005/118528 A2 published 15 Dec. 2005.-   Rincon, 2012 “Interleukin-6: from an inflammatory marker to a target    for inflammatory diseases”, Trends in Immunology, Vol. 33, No. 11,    pp. 571-577.-   Roodman, 2006, “Regulation of osteoclast differentiation”,    Ann. N. Y. Acad. Sci., Vol. 1068, pp. 100-109.-   Scott et al., 2010, “Rheumatoid Arthritis”, Lancet, Vol. 376, pp.    1094-1108.-   Sharif et al., 2012, “IRF5 polymorphism predicts prognosis in    patients with systemic sclerosis”, Annals of the Rheumatic Diseases,    Vol. 71, pp. 1197-1202.-   Smolen et al., 2003, “Therapeutic Strategies for Rheumatoid    Arthritis”, Nat. Rev. Drug Disc., Vol. 2, pp. 473-488.-   Steger et al., 2011, “Denosumab for the treatment of bone metastases    in breast cancer: evidence and opinion”, Ther. Adv. Med. Oncol.,    Vol. 3, pp. 233-243.-   Sugiyama et al., 1995, “Chemokines in bronchoalveolar lavage fluid    in summer-type hypersensitivity pneumonitis”, Eur. Respir. J., Vol.    8, pp. 1084-1090.-   Sun, 2010, “Mechanical loading, cartilage degradation and    arthritis”, Annals of the New York Academy of Sciences, Vol. 1211,    pp. 37-50.-   Takaoka et al., 2005, “Integral role of IRF-5 in the gene induction    programme activated by Toll-like receptors”, Nature, Vol. 434, pp.    243-249.-   Takayanagi, 2009, “Osteoimmunology and the effects of the immune    system on bone”, Nature Reviews Rheumatology, Vol. 5, pp. 667-676.-   Tanaka et al., 2003, “Signal transduction pathways regulating    osteoclast differentiation and function”, J. Bone Miner. Metab.,    Vol. 21, pp. 123-133.-   Tsutsumi et al., 2005, “Dipeptidyl peptidase IV inhibitor”,    international patent publication number WO 2005/025554 A2 published    24 Mar. 2005.-   van den Berg et al., 1999, “Pathogenesis of joint damage in    rheumatoid arthritis: evidence of a dominant role for    interleukin-I”, Baillieres Best Pract. Res. Clin. Rheumatol., Vol.    13, pp. 577-597.-   van den Berg, 2002, “Is there a rationale for combined TNF and IL-1    blocking in arthritis?”, Clin. Exp. Rheumatol., Vol. 20, pp.    S21-S25.-   Volejnikova et al., 1997, “Monocyte recruitment and expression of    monocyte chemoattractant protein-1 are developmentally regulated in    remodeling bone in the mouse”, Am. J. Pathol., Vol. 150, No. 5, pp.    1711-1721.-   Wang et al., 2010, “Selective ligands for the dopamine 3 (D₃)    receptor and methods of using same”, international patent    publication number WO 2010/025235 A1 published 4 Mar. 2010.-   Zhang et al., 2012 “Regulation of T helper cell differentiation by    interferon regulatory factor family members”, Immunol. Res., Vol. 54    pp. 169-176.-   Zheng et al., 1998, “Gene expression of monocyte chemoattractant    protein-1 in giant cell tumors of bone osteoclastoma: Possible    involvement in CD68⁺ macrophage-like cell migration”, Journal of    Cellular Biochemistry, Vol. 70, No. 1, pp. 121-129.

The invention claimed is:
 1. A method of treatment of rheumatoidarthritis comprising administering to a patient in need of treatment atherapeutically effective amount of a compound selected from compoundsof the following formulae, or a pharmaceutically acceptable saltthereof:


2. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


3. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


4. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


5. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


6. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


7. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


8. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


9. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


10. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


11. A method according to claim 1, wherein the compound is a compound ofthe following formula, or a pharmaceutically acceptable salt thereof:


12. A method of treatment of rheumatoid arthritis comprisingadministering to a patient in need of treatment a therapeuticallyeffective amount of a compound selected from compounds of the followingformulae, or a pharmaceutically acceptable salt thereof:


13. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


14. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


15. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


16. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


17. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


18. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


19. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


20. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


21. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


22. A method according to claim 12, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


23. A method of treatment of rheumatoid arthritis comprisingadministering to a patient in need of treatment a therapeuticallyeffective amount of a compound selected from compounds of the followingformulae, or a pharmaceutically acceptable salt thereof:


24. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


25. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


26. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


27. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


28. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


29. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


30. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


31. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


32. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof:


33. A method according to claim 23, wherein the compound is a compoundof the following formula, or a pharmaceutically acceptable salt thereof: